Nucleotide sequences for gene regulation and methods of use thereof

ABSTRACT

The invention provides nucleic acid sequences which regulate expression of a nucleotide sequence of interest. In particular, the invention provides nucleic acid sequences which regulate expression of a nucleotide sequence of interest in an age-related manner and/or in a liver-specific manner. The invention further provides methods of using the regulatory nucleic acid sequences provided herein for age-related and/or liver-specific expression of nucleotides seuqences of interest. The invention also provides host cells and transgenic non-human animals which harbor the regulatory nucleic acid sequences of the invention. The compositions and methods of the invention are useful in regulating expression of a nucleotide sequence of interest in an age-related and/or liver-specific manner.

This work was made, in part, with Government support by the NationalInstitutes of Health grant numbers HL38644 and HL53713. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

The invention relates to nucleic acid sequences which regulateexpression of a nucleotide sequence of interest. In particular, theinvention relates to nucleic acid sequences which regulate expression ofa nucleotide sequence of interest in an age-related manner and/or in aliver-specific manner. The invention further relates to methods of usingthe regulatory nucleic acid sequences provided herein for age-relatedand/or liver-specific expression of nucleotides sequences of interest.The invention also relates to host cells and to transgenic non-humananimals which harbor the regulatory nucleic acid sequences of theinvention. The compositions and methods of the invention are useful inregulating expression of a nucleotide sequence of interest in anage-related and/or liver-specific manner.

BACKGROUND OF THE INVENTION

A multitude of human diseases (e.g., thrombosis, cardiovasculardiseases, diabetes, Alzheimer's disease, cancer, osteoporosis,osteoarthritis, Parkinson's disease, dementia) are associated withincreasing age and result in serious effects on the quality of life andon the life expectancy of individuals suffering from such diseases.Other diseases (e.g., cirrhosis, primary and metastatic neoplasia,Wilson disease, hepachromatosis, infectious hepatitis, hepatic necrosis,Gilbert disease, Criggler-Najar disease) which afflict the liver alsohave serious clinical manifestations and are responsible for highmorbidity and mortality.

The treatment of age-related diseases (i.e., diseases whose prevalenceand/or severity of clinical manifestations increases with the age of thepatient) and diseases afflicting the liver focuses on the alleviation ofthe general symptoms of the disease using one or a combination of twomodalities, i.e., non-pharmacological treatment and pharmacologicaltreatment. Non-pharmacological treatment include, for example, periodsof bed rest and dietary changes. Non-pharmacological treatment is oftenused as an adjunct to pharmacological treatment which involves the useof drugs. Unfortunately, many of the commonly used pharmacologicalagents have numerous side effects and their use is further exacerbatedby the non-responsiveness by many patients with severe disease, who,paradoxically, are in most need of treatment. Both non-pharmacologicaland pharmacological treatments provide unsatisfactory approaches totreating age-related and liver-associated diseases because theseapproaches are often ineffective, their effects are inconsistent, andare directed to alleviating the general symptoms of disease, rather thanto specifically addressing the source of morbidity and mortality.Moreover, no suitable animal models are currently available torationally design drugs which target specific biochemical andphysiological pathways which are associated with age-related and withliver-associated diseases.

What is needed are methods for age-related and liver-specific geneexpression and models for age-related and liver-specific diseases.

SUMMARY OF THE INVENTION

The invention provides nucleic acid sequences which regulate expressionof a nucleotide sequence of interest in an age-related manner, as wellas nucleic acid sequences which direct liver-specific expression of agene of interest. Further provided by the invention are transgenicanimals which may be used as models for age-related and/or liverspecific diseases.

In one embodiment, the invention provides a recombinant expressionvector comprising in operable combination i) a nucleic acid sequence ofinterest, ii) a promoter sequence, and iii) one or more age regulatorysequences selected from SEQ ID NO:1, SEQ ID NO:3, a portion of SEQ IDNO:1, and a portion of SEQ ID NO:3. Without intending to limit theinvention to any particular type or source of nucleic acids sequence ofinterest, in a preferred embodiment, the nucleic acid sequence ofinterest encodes a protein selected from factor VIII, factor VII, factorIX, factor X, prothrombin, protein C, antithrombin III, tissue factorpathway inhibitor, LDL-receptor, human α1-antitrypsin, antithrombin III,PEA-3 protein, β-galactosidase, and luciferase. While it is not intendedthat the invention be restricted to any particular type or source ofpromoter sequence, in an alternative preferred embodiment, the promotersequence is selected from human factor IX promoter, cytomegaloviruspromoter, tRNA promoter, 5S rRNA promoters, histone gene promoters, RSVpromoter, retrovirus LTR promoter, SV40 promoter, PEPCK promoter, MTpromoter, SRα promoter, P450 family promoters, GAL7 promoter, T₇promoter, T₃ promoter, SP6 promoter, K11 promoter, and HIV promoter. Itis not contemplated that the invention be limited to any particular ageregulatory sequence which is a portion of SEQ ID NO:1. However, inanother preferred embodiment, the age regulatory sequence which is aportion of SEQ ID NO: 1 is selected from SEQ ID NO:2, SEQ ID NO:33, SEQID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38.Without intending to limit the invention to any particular rageregulatory sequence which s a portion of SEQ ID NO:3, in yet anotherpreferred embodiment, the age regulatory sequence which is a portion ofSEQ ID NO:3 is selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.

Also provided by the invention is a host cell containing a recombinantexpression vector comprising in operable combination i) a nucleic acidsequence of interest, ii) a promoter sequence, and iii) one or more ageregulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portionof SEQ ID NO:1, and a portion of SEQ ID NO:3. Without intending to limitthe invention to the environment in which the host cell is contained, inone preferred embodiment, the host cell is comprised in a tissue ororgan in a living animal. In another preferred embodiment, the host cellis a gamete. In yet another preferred embodiment, the host cell isselected from bacterial cell, yeast cell, plant cell, insect cell, andmammalian cell.

The invention also provides a recombinant expression vector comprisingin operable combination i) a nucleic acid sequence of interest, ii) apromoter sequence, and iii) a functional homolog of one or more ageregulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3, a portionof SEQ ID NO:1, and a portion of SEQ ID NO:3. Without limiting theinvention to the type or source of the nucleic acid sequence ofinterest, in one preferred embodiment, the nucleic acid sequence ofinterest encodes a protein selected from factor VIII, factor VII, factorIX, factor X, prothrombin, protein C, antithrombin III, tissue factorpathway inhibitor, LDL-receptor, human α1-antitrypsin, antithrombin III,PEA-3 protein, β-galactosidase, and luciferase. While it is not intendedthat the invention be limited to the type or source of the promotersequence, in an alternative preferred embodiment, the promoter sequenceis selected from human factor IX promoter, cytomegalovirus promoter,tRNA promoter, 5S rRNA promoters, histone gene promoters, RSV promoter,retrovirus LTR promoter, SV40 promoter, PEPCK promoter, MT promoter, SRαpromoter, P450 family promoters, GAL7 promoter, T₇ promoter, T₃promoter, SP6 promoter, K11 promoter, and HIV promoter. Though it is notcontemplated that the invention be limited to the portion of SEQ ID NO:1which has age-related regulatory activity, in another preferredembodiment, the age regulatory sequence which is a portion of SEQ IDNO:1 is selected from SEQ ID NO:2, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. Without intendingto limit the invention the portion of SEQ ID NO:3 which has age-relatedregulatory activity, in yet another preferred embodiment, the ageregulatory sequence which is a portion of SEQ ID NO:3 is selected fromSEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60,and SEQ ID NO:61.

Also provided herein is a host cell containing recombinant expressionvector comprising in operable combination i) a nucleic acid sequence ofinterest, ii) a promoter sequence, and iii) a functional homolog of oneor more age regulatory sequences selected from SEQ ID NO:1, SEQ ID NO:3,a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3. Withoutintending to limit the invention to the environment in which the hostcell is contained, in one preferred embodiment, the host cell iscomprised in a tissue or organ in a living animal. In an alternativepreferred embodiment, the host cell is a gamete. In another preferredembodiment, the host cell is selected from bacterial cell, yeast cell,plant cell, insect cell, and mammalian cell.

The invention also provides a method, comprising: a) providing: i) acell, ii) a nucleic acid sequence of interest, iii) a promoter sequence,and iv) one or more age regulatory sequences selected from SEQ ID NO:1,SEQ ID NO:3, a portion of SEQ ID NO:1, and a portion of SEQ ID NO:3; b)operably linking the nucleic acid sequence of interest, the promotersequence, and the one or more age regulatory sequences to produce atransgene; and c) introducing the transgene into the cell to create atreated cell under conditions such that the nucleic acid sequence ofinterest is expressed in the treated cell. Without intending to limitthe treated cell to any particular environment, in one preferredembodiment, the treated cell is comprised in a tissue or organ in aliving animal.

The invention further provides a substantially purified nucleic acidsequence comprising a nucleotide sequence selected from a functionalhomolog of SEQ ID NO:1 and of the complement thereof.

Also provided herein is a substantially purified nucleic acid sequencecomprising a nucleotide sequence selected from a functional homolog ofSEQ ID NO:3 and of the complement thereof.

Also provided by the present invention is a substantially purifiednucleic acid sequence comprising a portion of a nucleotide sequenceselected from a functional homolog of SEQ ID NO:1 and of the complementthereof. In one embodiment, the portion is SEQ ID NO:2. In analternative embodiment, the portion is selected from SEQ ID NO:33, SEQID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.

The invention also provides a substantially purified nucleic acidsequence comprising a portion of a nucleotide sequence selected from afunctional homolog of SEQ ID NO:3 and of the complement thereof. In oneembodiment, the portion is selected from SEQ ID NO:51, SEQ ID NO:52, SEQID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.

Also provided herein is a substantially purified nucleic acid sequencewhich hybridizes under stringent hybridization conditions with SEQ IDNO:1 or with the complement thereof, wherein the nucleic acid sequenceis characterized by having age-related regulatory activity, and byhaving greater than 63% and less than 100% homology to the SEQ ID NO:1.

The invention also provides a substantially purified nucleic acidsequence which hybridizes under stringent hybridization conditions withSEQ ID NO:3 or with the complement thereof, wherein the nucleic acidsequence is characterized by having age-related regulatory activity, andby having greater than 60% and less than 100% homology to the SEQ IDNO:3.

The invention additionally provides a recombinant expression vectorcomprising at least a portion of a nucleotide sequence selected from afunctional homolog of SEQ ID NO:1 and of the complement thereof.

Also provided herein is a recombinant expression vector comprising atleast a portion of a nucleotide sequence selected from a functionalhomolog of SEQ ID NO:3 and of the complement thereof.

The invention also provides a transgenic cell comprising at least aportion of a nucleotide sequence selected from a functional homolog ofSEQ ID NO:1 and of the complement thereof. In one embodiment, thenucleotide sequence is operably linked to a promoter and to a nucleicacid sequence of interest. In a preferred embodiment, the transgeniccell is comprised in an animal. In a more preferred embodiment, thenucleic acid sequence of interest is expressed in an age-related mannerin the transgenic cell.

The invention additionally provides a transgenic cell comprising atleast a portion of a nucleotide sequence selected from a functionalhomolog of SEQ ID NO:3 and of the complement thereof. In one embodiment,the nucleotide sequence is operably linked to a promoter and to anucleic acid sequence of interest. In a preferred embodiment, thetransgenic cell is comprised in an animal. In a more preferredembodiment, the nucleic acid sequence of interest is expressed in anage-related manner in the transgenic cell.

The invention also provides a method for expressing a nucleic acidsequence of interest in a cell, comprising: a) providing: i) a cell; ii)a nucleic acid sequence of interest; iii) a promoter sequence; iv) SEQID NO:1; and v) SEQ ID NO:3; b) operably linking the nucleic acidsequence of interest, the promoter sequence, the SEQ ID NO:1 and the SEQID NO:3 to produce a transgene; and c) introducing the transgene intothe cell to produce a transgenic cell under conditions such that thenucleic acid sequence of interest is expressed in the transgenic cell.In one embodiment, the cell expresses a recombinant protein identifiedas SEQ ID NO:47. In an alternative embodiment, the cell is selected fromHepG2 cell, fibroblast cell, myoblast cell, and endothelial cell. Inanother embodiment, the cell is a fertilized egg cell, and thetransgenic cell is a transgenic fertilized egg cell. In a preferredembodiment, the method further comprises d) introducing the transgenicfertilized egg cell into a non-human animal and permitting the animal todeliver progeny containing the transgene. In a more preferredembodiment, the progeny is characterized by age-related expression ofthe nucleic acid sequence of interest. In an alternative more preferredembodiment, the progeny is characterized by liver-specific expression ofthe nucleic acid sequence of interest. In another preferred embodiment,the fertilized egg cell is derived from a mammal of the order Rodentia.In a more preferred embodiment, the fertilized egg cell is a mousefertilized egg cell. In yet another embodiment, the promoter is selectedfrom human factor IX promoter, cytomegalovirus promoter, tRNA promoter,5S rRNA promoters, histone gene promoters, RSV promoter, retrovirus LTRpromoter, SV40 promoter, PEPCK promoter, MT promoter, SRα promoter, P450family promoters, GAL7 promoter, T₇ promoter, T₃ promoter, SP6 promoter,K11 promoter, and HIV promoter. In a further embodiment, the nucleicacid sequence of interest encodes a protein selected from factor VIII,factor VII, factor IX, factor X, prothrombin, protein C, antithrombinIII, tissue factor pathway inhibitor, LDL-receptor, humanα1-antitrypsin, antithrombin III, PEA-3 protein, β-galactosidase, andluciferase.

The invention also provides a method for expressing a nucleic acidsequence of interest in a cell, comprising: a) providing: i) a cell; ii)a nucleic acid sequence of interest; iii) a promoter sequence; iv) aportion of SEQ ID NO:1; and v) a portion of SEQ ID NO:3; b) operablylinking the nucleic acid sequence of interest, the promoter sequence,the portion of SEQ ID NO:1 and the portion of SEQ ID NO:3 to produce atransgene; and c) introducing the transgene into the cell to produce atransgenic cell under conditions such that the nucleic acid sequence ofinterest is expressed in the transgenic cell.

Additionally provided by the invention is a method for expressing anucleic acid sequence of interest in a cell, comprising: a) providing:i) a cell; ii) a nucleic acid sequence of interest; iii) a promotersequence; and iv) SEQ ID NO:1; b) operably linking the nucleic acidsequence of interest, the promoter sequence, and the SEQ ID NO:1 toproduce a transgene; and c) introducing the transgene into the cell toproduce a transgenic cell under conditions such that the nucleic acidsequence of interest is expressed in the transgenic cell.

Also provided herein is a method for expressing a nucleic acid sequenceof interest in a cell, comprising: a) providing: i) a cell; ii) anucleic acid sequence of interest; iii) a promoter sequence; and iv) aportion of SEQ ID NO:1; b) operably linking the nucleic acid sequence ofinterest, the promoter sequence, and the portion of SEQ ID NO:1 toproduce a transgene; and c) introducing the transgene into the cell toproduce a transgenic cell under conditions such that the nucleic acidsequence of interest is expressed in the transgenic cell.

The invention further provides a method for expressing a nucleic acidsequence of interest in a cell, comprising: a) providing: i) a cell; ii)a nucleic acid sequence of interest; iii) a promoter sequence; and iv)SEQ ID NO:3; b) operably linking the nucleic acid sequence of interest,the promoter sequence, and the SEQ ID NO:3 to produce a transgene; andc) introducing the transgene into the cell to produce a transgenic cellunder conditions such that the nucleic acid sequence of interest isexpressed in the transgenic cell.

Further provided by the invention is a method for expressing a nucleicacid sequence of interest in a cell, comprising: a) providing: i) acell; ii) a nucleic acid sequence of interest; iii) a promoter sequence;and iv) a portion of SEQ ID NO:3; b) operably linking the nucleic acidsequence of interest, the promoter sequence, and the portion of SEQ IDNO:3 to produce a transgene; and c) introducing the transgene into thecell to produce a transgenic cell under conditions such that the nucleicacid sequence of interest is expressed in the transgenic cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of eleven exemplary human FIX minigeneexpression constructs and relative in vitro transient expressionactivities (ng hFIX/10⁶ cells/48 hr).

FIG. 2 shows graphs of longitudinal analyses of transgenic mice whichcarry −416FIXm1 (A), −416FIXm1/1.4 (B), −590FIXm1 (C), −679FIXm1 (D),and −770FIXm1 (E) expression vectors and which produce high initialprepubertal, but rapidly decreasing, hFIX expression levels with age.

FIG. 3 shows a Northern blot of human FIX mRNA levels (A) and a gelshowing hFIX transgene DNA levels as determined by multiplex PCRanalysis (B) in the livers and tails of animals carrying −416FIXm1.

FIG. 4 shows graphs of longitudinal analysis of transgenic mice whichcarry −802FIXm1 (A), −802FIXm1/1.4 (B), −2231FIXm1 (C), −2231FIXm1/1.4(D) and −416FIXm1/AE5′ (E) expression vectors and which produce hFIX atstable and increasing levels with age.

FIG. 5 shows a Northern blot of transgenic mice carrying −802FIXm1 and−802FIXm1/1.4 expression vectors.

FIG. 6 is a gel of a gel electrophoretic mobility shift assay usingmouse liver nuclear extract (NEs) from three different age groups, andusing double-stranded oligonucleotides containing a PEA-3 nucleotidesequence spanning from nt −797 to −776 of the hFIX gene (A), and using acompetition assay for ³²P-labelled double stranded oligonucleotidescontaining the PEA-3 nucleotide sequence (B).

FIG. 7 is a Northern blot showing tissue specificity of hFIX expressionin transgenic mice carrying −416FIXm1 (A) and −802 FIXm1 (B) expressionvectors.

FIG. 8A-E shows the nucleotide sequence (SEQ ID NO:4) of, and eightamino acid sequences (SEQ ID NOs:5 to 12) which together form, the humanfactor IX (GenBank accession number K02402). The initiationtranscription site (nucleotide 1) and the poly-A addition site(nucleotide 32,757) are identified by solid circles. The solid verticalarrows indicate the intron-exon splice junction. The five Alu repetitivesequences have been underlined, while the 5-base insert in intron A andthe AATAAA sequence in exon VIII are boxed. The cleavage or terminationsite at the 3′ end of the gene (CATTG) is underlined with a dashed line.

FIG. 9 shows the cDNA sequence (SEQ ID NO:13) (A) and encodedpolypeptide sequence (SEQ ID NO:47) (B) of mouse PEA-3 (GenBankaccession number X63190).

FIG. 10 A-D shows the cDNA sequence (SEQ ID NO:42) of the humanα1-antitrypsin gene (GenBank accession number K02212).

FIG. 11 shows the DNA sequence (SEQ ID NO:43) of human antithrombin III(GenBank accession number A06100).

FIG. 12 shows the cDNA sequence (A) (SEQ ID NO:49) (GenBank accessionnumber X02750) and genomic DNA sequence (B) (SEQ ID NO:50) (GenBankaccession number M11228) of human protein C.

FIG. 13 (A-E) shows the nucleic acid sequences (SEQ ID NOs:76-83) ofexemplary homologs of AE3′ (SEQ ID NO:3).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated nucleic acid sequence” refers to a nucleic acid sequence thatis identified and separated from at least one contaminant nucleic acidwith which it is ordinarily associated in its natural state, or whenobtained from its actual source. Isolated nucleic acid is nucleic acidpresent in a form or setting that is different from that in which it isfound in nature. In contrast, non-isolated nucleic acids are nucleicacids such as DNA and RNA which are found in the state they exist innature. For example, a given DNA sequence (e.g., a gene) is found on thehost cell chromosome in proximity to neighboring genes; RNA sequences,such as a specific mRNA sequence encoding a specific protein, are foundin the cell as a mixture with numerous other mRNAs which encode amultitude of proteins. However, an isolated nucleic acid sequencecomprising SEQ ID NO:1 includes, by way of example, such nucleic acidsequences in cells which ordinarily contain SEQ ID NO:1 where thenucleic acid sequence is in a chromosomal or extrachromosomal locationdifferent from that of natural cells, or is otherwise flanked by adifferent nucleic acid sequence than that found in nature. The isolatednucleic acid sequence may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid sequence is to beutilized to express a protein, the nucleic acid sequence will contain(at a minimum) at least a portion of the sense or coding strand (i.e.,the nucleic acid sequence may be single-stranded). Alternatively, it maycontain both the sense and anti-sense strands (i.e., the nucleic acidsequence may be double-stranded).

As used herein, the term “purified” refers to molecules, either nucleicor amino acid sequences, that are removed from their naturalenvironment, isolated or separated. An “isolated nucleic acid sequence”is therefore a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated.

The term “recombinant” when made in reference to a DNA sequence refersto a DNA sequence which is comprised of segments of DNA joined togetherby means of molecular biological techniques. The term “recombinant” whenmade in reference to a polypeptide sequence refers to a polypeptidesequence which is expressed using a recombinant DNA sequence.

As used herein, the terms “vector” and “vehicle” are usedinterchangeably in reference to nucleic acid molecules that transfer DNAsegment(s) from one cell to another.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

The term “transgenic” when used in reference to a cell refers to a cellwhich contains a transgene, or whose genome has been altered by theintroduction of a transgene. The term “transgenic” when used inreference to a tissue or animal refers to a tissue or animal,respectively, which comprises one or more cells that contain atransgene, or whose genome has been altered by the introduction of atransgene. Transgenic cells, tissues and animals may be produced byseveral methods including the introduction of a “transgene” comprisingnucleic acid (usually DNA) into a target cell or integration of thetransgene into a chromosome of a target cell by way of humanintervention, such as by the methods described herein.

A “non-human animal” refers to any animal which is not a human andincludes vertebrates such as rodents, non-human primates, ovines,bovines, ruminants, lagomorphs, porcines, caprines, equines, canines,felines, aves, etc. Preferred non-human animals are selected from theorder Rodentia. The term “order Rodentia” refers to rodents i.e.,placental mammals (class Euthria) which include the family Muridae(e.g., rats and mice), most preferably mice.

The term “nucleotide sequence of interest” refers to any nucleotidesequence, the manipulation of which may be deemed desirable for anyreason (e.g., treat disease, confer improved qualities, etc.), by one ofordinary skill in the art. Such nucleotide sequences include, but arenot limited to, coding sequences of structural genes (e.g., reportergenes, selection marker genes, oncogenes, drug resistance genes, growthfactors, etc.), and non-coding regulatory sequences which do not encodean mRNA or protein product (e.g., promoter sequence, polyadenylationsequence, termination sequence, enhancer sequence, etc.).

As used herein, the terms “complementarity,” or “complementary” are usedin reference to nucleotide sequences related by the base-pairing rules.For example, the sequence 5′-AGT-3′ is complementary to the sequence5′-ACT-3′. Complementarity can be “partial” or “total.” “Partial”complementarity is where one or more nucleic acid bases is not matchedaccording to the base pairing rules. “Total” or “complete”complementarity between nucleic acids is where each and every nucleicacid base is matched with another base under the base pairing rules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands.

A “complement” of a nucleic acid sequence as used herein refers to anucleotide sequence whose nucleic acids show total complementarity tothe nucleic acids of the nucleic acid sequence.

The term “homology” when used in relation to nucleic acids refers to adegree of complementarity. There may be partial homology (i.e., partialidentity) or complete homology (i.e., complete identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid sequence and is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe (i.e., an oligonucleotidewhich is capable of hybridizing to another oligonucleotide of interest)will compete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence to a target sequence under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target which lacks even a partialdegree of complementarity (e.g., less than about 30% identity); in theabsence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described infra.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe which can hybridizeto the single-stranded nucleic acid sequence under conditions of lowstringency as described infra.

The term “hybridization” as used herein includes “any process by which astrand of nucleic acid joins with a complementary strand through basepairing.” [Coombs J (1994) Dictionary of Biotechnology, Stockton Press,New York N.Y.]. Hybridization and the strength of hybridization (i.e.,the strength of the association between the nucleic acids) is impactedby such factors as the degree of complementarity between the nucleicacids, stringency of the conditions involved, the T_(m) of the formedhybrid, and the G:C ratio within the nucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl [see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)]. Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

Low stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 68° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 1% SDS, 5×Denhardt's reagent [50× Denhardt's contains the following per 500 ml: 5g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 0.2×SSPE, and 0.1% SDS at room temperature when a DNA probeof about 100 to about 1000 nucleotides in length is employed.

High stringency conditions when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5× Denhardt'sreagent and 100 μg/ml denatured salmon sperm DNA followed by washing ina solution comprising 0.1×SSPE, and 0.1% SDS at 68° C. when a probe ofabout 100 to about 1000 nucleotides in length is employed.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 50% to 70%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with other nucleic acidsequences that have from 50% to 70% homology to the first nucleic acidsequence.

When used in reference to nucleic acid hybridization the art knows wellthat numerous equivalent conditions may be employed to comprise eitherlow or high stringency conditions; factors such as the length and nature(DNA, RNA, base composition) of the probe and nature of the target (DNA,RNA, base composition, present in solution or immobilized, etc.) and theconcentration of the salts and other components (e.g., the presence orabsence of formamide, dextran sulfate, polyethylene glycol) areconsidered and the hybridization solution may be varied to generateconditions of either low or high stringency hybridization differentfrom, but equivalent to, the above-listed conditions.

Those skilled in the art know that whereas higher stringencies may bepreferred to reduce or eliminate non-specific binding of the nucleotidesequence of SEQ ID NOs:1 or 3 with other nucleic acid sequences, lowerstringencies may be preferred to detect a larger number of nucleic acidsequences having different homologies to the nucleotide sequence of SEQID NOs:1 and 3.

As used herein, the terms “regulatory element” and “regulatory sequence”interchangeably refer to a nucleotide sequence which does not encode RNAor a protein and which controls some aspect of the expression of nucleicacid sequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. In contrast, the term “regulatorygene” refers to a DNA sequence which encodes RNA or a protein (e.g.,transcription factor) that controls the expression of other genes.

Regulatory elements may be tissue specific or cell specific. The term“tissue specific” as it applies to a regulatory element refers to aregulatory element that is capable of directing selective expression ofa nucleotide sequence of interest to a specific type of tissue (e.g.,liver) in the relative absence of expression of the same nucleotidesequence of interest in a different type of tissue (e.g., lung).

Tissue specificity of a regulatory element may be evaluated by, forexample, operably linking a reporter gene to a promoter sequence (whichis not tissue-specific) and to the regulatory element to generate areporter construct, introducing the reporter construct into the genomeof an animal such that the reporter construct is integrated into everytissue of the resulting transgenic animal, and detecting the expressionof the reporter gene (e.g., detecting mRNA, protein, or the activity ofa protein encoded by the reporter gene) in different tissues of thetransgenic animal. The detection of a greater level of expression of thereporter gene in one or more tissues relative to the level of expressionof the reporter gene in other tissues shows that the regulatory elementis “specific” for the tissues in which greater levels of expression aredetected. Thus, the term “tissue-specific” (e.g., liver-specific) asused herein is a relative term that does not require absolutespecificity of expression. In other words, the term “tissue-specific”does not require that one tissue have extremely high levels ofexpression and another tissue have no expression. It is sufficient thatexpression is greater in one tissue than another. By contrast, “strict”or “absolute” tissue-specific expression is meant to indicate expressionin a single tissue type (e.g., liver) with no detectable expression inother tissues.

The term “cell type specific” as applied to a regulatory element refersto a regulatory element which is capable of directing selectiveexpression of a nucleotide sequence of interest in a specific type ofcell in the relative absence of expression of the same nucleotidesequence of interest in a different type of cell within the same tissue.The term “cell type specific” when applied to a regulatory element alsomeans a regulatory element capable of promoting selective expression ofa nucleotide sequence of interest in a region within a single tissue.

Cell type specificity of a regulatory element may be assessed usingmethods well known in the art, e.g., immunohistochemical staining and/orNorthern blot analysis. Briefly, for immunohistochemical staining,tissue sections are embedded in paraffin, and paraffin sections arereacted with a primary antibody which is specific for the polypeptideproduct encoded by the nucleotide sequence of interest whose expressionis regulated by the regulatory element. A labeled (e.g., peroxidaseconjugated) secondary antibody which is specific for the primaryantibody is allowed to bind to the sectioned tissue and specific bindingdetected (e.g., with avidin/biotin) by microscopy. Briefly, for Northernblot analysis, RNA is isolated from cells and electrophoresed on agarosegels to fractionate the RNA according to size followed by transfer ofthe RNA from the gel to a solid support, such as nitrocellulose or anylon membrane. The immobilized RNA is then probed with a labeledoligo-deoxyribonucleotide probe or DNA probe to detect RNA speciescomplementary to the probe used. Northern blots are a standard tool ofmolecular biologists.

The term “promoter,” “promoter element,” or “promoter sequence” as usedherein, refers to a DNA sequence which when ligated to a nucleotidesequence of interest is capable of controlling the transcription of thenucleotide sequence of interest into mRNA. A promoter is typically,though not necessarily, located 5′ (i.e., upstream) of a nucleotidesequence of interest whose transcription into mRNA it controls, andprovides a site for specific binding by RNA polymerase and othertranscription factors for initiation of transcription.

Promoters may be constitutive or regulatable. The term “constitutive”when made in reference to a promoter means that the promoter is capableof directing transcription of an operably linked nucleic acid sequencein the absence of a stimulus (e.g., heat shock, chemicals, etc.). Incontrast, a “regulatable” promoter is one which is capable of directinga level of transcription of an operably linked nucleic acid sequence inthe presence of a stimulus (e.g., heat shock, chemicals, etc.) which isdifferent from the level of transcription of the operably linked nucleicacid sequence in the absence of the stimulus.

DESCRIPTION OF THE INVENTION

The invention provides nucleic acid sequences which regulate expressionof a nucleotide sequence of interest. In on embodiment, the inventionprovides nucleic acid sequences which regulate expression of anucleotide sequence of interest in an age-related manner. Yet moreparticularly, the exemplary age-regulatory element 5′ (AE5′) has beendiscovered to regulate stable gene expression over time in vivo, whilethe exemplary age-regulatory element 3′ (AE3′) has been discovered toregulate increased gene expression over time in vivo. In anotherembodiment, the invention provides nucleic acid sequences (e.g., AE5′)which direct liver-specific expression of a gene of interest. In yetanother embodiment, the invention provides transgenic animals whichharbor the nucleic acid sequences provided herein and which expres anucleotide sequence of interest in an age-related and/or liver-specificmanner. The nucleic acid sequences provided herein are useful in, forexample, identifying and isolating functional homologs of AE5′ and AE3′,and amplifying at least a portion of AE5′ and AE3′. Importantly, thenucleic acid sequences of the invention are also useful in age-relatedexpression and/or liver-specific expression of a nucleotide sequence ofinterest in an animal, in gene therapy, and in reducing expression offactor IX in an animal.

The invention is further discussed under (A) Regulatory Nucleic AcidSequences, (B) Using Probes To Identify And Isolate Homologs Of AE5′ andAE3′, (C) Using Primers to Amplify At Least A Portion Of AE5′ And AE3′,(D) Methods For Regulating Gene Expression, (E) Gene Therapy, and (F)Reducing Expression Of Factor IX In An Animal.

A. Regulatory Nucleic Acid Sequences

The regulatory nucleic acid sequences of the invention and theirsurprising properties in regulating gene expression were discoveredduring the inventor's investigation of the mechanisms underlyingage-associated regulation of the human factor IX, which is involved inblood coagulation. Blood coagulation plays a critical role not only inhomeostasis, but also in many physiological and pathological conditions[Saito in Disorders of Hemostasis, O. D. Ratnoff and C. D. Forbes, Eds.,Sauders, Philadelphia, ed. 2 (1991), pp. 18-47; Kurachi et al. (1993)Blood Coagul. Fibrinol. 4:953-974]. Blood coagulation potential inhumans as well as in other mammals reaches the young adult level aroundthe age of weaning [Yao et al. (1991) Thromb. Haemost. 65:52-58; Andrewet al. (1992) Blood 80:1998-2005; Andrew et al. Blood (1987) 70:165-172;Andrew et al. (1988) Blood 72:1651-1657]. This is followed by a gradualincrease in coagulation potential during young adulthood, and an almosttwo-fold increase by old age [Sweeney and Hoemig (1993) Am. J. Clin.Pathol. 99:687-688; Kurachi et al. (1996) Thromb. Haemost. 76:965-969].This age-associated increase in coagulation potential takes place inhealthy centenarians [Marie et al. (1995) Blood 85:3144-3149],indicating that the increase is a normal phenomenon associated withaging.

It is the inventors' consideration that this increase in coagulationpotential may make a crucial contribution to the development andprogression of age-associated diseases such as cardiovascular andthrombotic disorders [Conlan et al. (1993) The Atherosclerosis Risk inCommunities (ARIC) Study 70:380-385; Balleisen et al. (1985) Thromb.Haemost. 54:475-479; Rode et al. (1996) Nat. Med. 2:293-298; Woodward etal. (1997) Brit. J. Haemat. 97:785-797]. The inventors' considerationwas based on the observation that this increase in blood coagulationpotential coincides with plasma level increases of pro-coagulant factorssuch as factor IX, whereas plasma levels of anti-coagulation factors(such as antithrombin III and protein C) or of factors involved infibrinolysis are only marginally affected [Conlan et al. (1994) TheAtherosclerosis Risk in Committees (ARIC) Study 72:551-556; Lowe et al.(1997) Brit. J. Haemat. 97:775-784]. These facts strongly suggested tothe inventors that the observed increase in blood coagulation activitywith advancing age is due to regulated events. Plasma levels of eachprotein factor involved in blood coagulation, fibrinolysis and theirregulatory systems are presumably determined by the balance of the manyprocesses involved. At present, little is known about why an advancingage-associated increase in blood coagulation activity exists, or whatmolecular mechanisms are involved in age-dependent regulation(homeostasis) of blood coagulation [Finch in Longevity, Senescence, andthe Genome, The University of Chicago Press, Chicago, 1990].

Blood coagulation factor IX (FIX) occupies a key position in the bloodcoagulation cascade where the intrinsic and extrinsic pathways merge[Saito in Disorders of Hemostasis, O. D. Ratnoff and C. D. Forbes, Eds.,Sauders, Philadelphia, ed. 2 (1991), pp. 18-47; Kurachi et al. (1993)Blood Coagul. Fibrinol. 4:953-974]. FIX is synthesized in the liver withstrict tissue-specificity, and its deficiency results in the bleedingdisorder hemophilia B. In normal humans the plasma activity and proteinconcentration levels of human FIX (hFIX) increase with advancing age[Sweeney and Hoemig (1993) Am. J. Clin. Pathol. 99:687-688; Kurachi etal. (1996) Thromb. Haemost. 76:965-969]. Mouse FIX (mFIX) plasmaactivity also increases with age in a manner similar to hFIX, and isdirectly correlated with an increase in liver mFIX messenger RNA (mRNA)level [Sweeney and Hoemig (1993) Am. J. Clin. Pathol. 99:687-688;Kurachi et al. (1996) Thromb. Haemost. 76:965-969]. However, nothingelse is known about the molecular mechanisms underlying such anincrease. In investigating the basic molecular mechanisms responsiblefor age-associated regulation of hFIX, the inventors discovered thenucleotide sequences which regulate age-associated expression, and whichdirect liver-specific expression, of the exemplary hFIX gene.

The discovery of the invention sequences was made possible, in part, bythe inventors' use of the hFIX promoter in combination with the codingsequence for hFIX instead of with the coding sequence for commonly usedreporter proteins. The discovery of the surprising functions of thenucleotide sequences provided herein was also made possible by theinventors' use of longitudinal in vivo analyses, rather than of in vitroanalyses. In particular, the inventors' earlier studies used reportergenes (including bacterial β-galactosidase and chloramphenicolacetyltransferase [CAT]) which are heterologous to the factor IXpromoter. In these earlier studies, the factor IX promoter showed onlyvery weak expression activity in vitro [Kurachi et al. (1995) J. Biol.Chem. 270:5276-5281]. Use of such heterologous reporter genes made itimpossible to reliably and quantitatively perform longitudinal analysesof transgene expression in animals. The inventors unexpectedly observedthat the use of hFIX minigene expression vectors which contained thehFIX promoter and its homologous hFIX gene were capable of producinghigh level plasma hFIX in vivo. This unexpected observation not onlysolved the problems associated with the use of genes which areheterologous to the hFIX promoter by providing a reliable animal assaysystem, but also provided multiple unexpected critical insights into theregulatory mechanisms of the hFIX gene, including the determination ofnucleotide sequences which regulate the stability and age-relatedincreased expression of the exemplary hFIX gene.

The present invention provides the 32-nucleotide nucleic acid sequence(SEQ ID NO:1) of AE5′ which corresponds to the sequence from 2164 to2195 of the hFIX gene deposited in GenBank as accession number K02402,and which corresponds to the sequence from −802 to −771 of GenBankaccession number K02402 when in relation to the hFIX start codon (ATG)in which the adenine is designated as position +30.

The present invention also provides the 1273-nucleotide nucleic acidsequence (SEQ ID NO:3) (FIG. 13) of AE3′ which corresponds to thesequence from 34,383 to 35,655 of GenBank accession number K02402, andwhich corresponds to the sequence from 31,418 to 32,690 of FIG. 8 whenin relation to the hFIX start codon (ATG) in which the adenine isdesignated as position zero.

The terms “age-related regulatory activity” and “age-related activity”when made in reference to a nucleic acid sequence refer to the abilityof the nucleic acid sequence to alter in an age-related manner (e.g.,increase over a period of time) the level of transcription into mRNAand/or the synthesis of a polypeptide encoded by nucleotide sequence ofinterest which is operably linked to a promoter sequence as compared tothe level of transcription into mRNA of the nucleotide sequence ofinterest which is operably linked to the promoter sequence in theabsence of the nucleic acid sequence which has age-related regulatoryactivity. An “age regulatory sequence” is herein used to refer to anucleic acid sequence which has age-related regulatory activity.

To illustrate, where expression levels of a gene of interest decreaseover a period of time, a nucleic acid sequence is said to haveage-related regulatory activity if (when operably linked to the gene ofinterest) it results in (a) a smaller decrease in expression levels ofthe gene over the same period of time as compared to the decrease inexpression levels in the absence of the nucleic acid sequence, (b)relatively constant (i.e., unchanged) expression levels over the sameperiod of time, or (c) increased expression levels over the same periodof time.

The terms “operably linked,” “in operable combination,” and “in operableorder” as used herein refer to the linkage of nucleic acid sequencessuch that they perform their intended function. For example, operablylinking a promoter sequence to a nucleotide sequence of interest refersto linking the promoter sequence and the nucleotide sequence of interestin a manner such that the promoter sequence is capable of directing thetranscription of the nucleotide sequence of interest and/or thesynthesis of a polypeptide encoded by the nucleotide sequence ofinterest. Similarly, operably linking a nucleic acid sequence havingage-related regulatory activity to a promoter sequence and to anucleotide sequence of interest means linking the nucleic acid sequencehaving age-related regulatory activity, the promoter sequence and thenucleotide sequence of interest in a manner such that the nucleic acidsequence having age-related regulatory activity is capable of alteringover a period of time the level of transcription into mRNA of thenucleotide sequence of interest and/or the synthesis of a polypeptideencoded by the nucleotide sequence of interest.

Methods for determining age-related regulatory activity of a candidatenucleic acid sequence, given the teachings of the present specification,are within the ordinary skill in the art and are exemplified by themethods disclosed herein. For example, a test vector is constructed inwhich the candidate nucleic acid sequence is linked upstream ordownstream of a promoter sequence which is operably linked to anucleotide sequence of interest (e.g., Example 1). A control vectorwhich is similar to the test vector but which lacks the candidatenucleic acid sequence is also constructed. The test vector and controlvector are separately introduced into a host cell. It is preferred thatthe host cell (e.g., fertilized egg) be capable of generating atransgenic multicellular organism, e.g., a transgenic mouse (e.g.,Example 3) and that transgenic multicellular organisms are generated.Longitudinal analyses of the expression of mRNA which is encoded by thenucleotide sequence of interest (e.g., by Northern blot hybridization)over a period of time in, and preferably over the entire life span of,founders and successive generations of the transgenic multicellularorganism are carried out (e.g., Example 3). The detection in any tissueof mRNA and/or protein levels which are encoded by the nucleotidesequence of interest and which are greater in transgenic animalsharboring the test vector as compared to the mRNA and/or protein levelsin transgenic animals harboring the control vector at least one point intime indicates that the candidate nucleic acid sequence has age-relatedregulatory activity.

For example, evidence provided herein shows the surprising result thatAE5′ (SEQ ID NO:1) alone has age-related regulatory activity in thatAE5′ stabilizes hFIX mRNA whereby hFIX mRNA levels are essentiallyunchanged at different time points over the entire life span oftransgenic animals (FIG. 4, A, C and E) as compared to the declininghFIX mRNA levels in transgenic animals which harbor vectors that lackAE5′ (FIGS. 2A and 2E). The age-related regulatory activity of AE5′ wasobserved regardless whether AE5′ was placed upstream (FIG. 4A) ordownstream (FIG. 4E) of the promoter sequence in the expressionconstruct.

Furthermore, data provided herein demonstrates the unexpected resultthat AE3′ (SEQ ID NO:3) alone has age-related regulatory activity inthat AE3′ increases hFIX mRNA at several time points during the life oftransgenic animals (FIG. 2B) relative to the hFIX mRNA levels at thesame time points in transgenic animals harboring vectors that lack AE3′(FIG. 2A). AE3′ substantially increased the steady state hFIX mRNAlevels (FIG. 5). This result which was observed in vivo was surprisingin part because AE3′ exhibited weak down regulatory effects on hFIXproduction in vitro. Without limiting the invention to any particularmechanism, these results suggest that AE3′ functions by increasing hFIXmRNA stability which directly correlates with an increase in the hFIXprotein level in the circulation. Also without intending to limit theinvention to any particular theory, it is the inventors' considerationthat the age-related regulatory activity of AE3′ is due to the slstructure-forming dinucleotide repeats present in the 3′UTR; the slregion is the 103 bp sequence (SEQ ID NO:61) from nt 32,141 through nt32,243 of FIG. 8. This consideration is based on the inventors'observation that dinucleotide repeats, such as (AT)_(n) of the 3′ UTR ofvarious genes, can form sl structures in mRNA, which upon bindingspecific proteins are known to modulate mRNA stability, mostly to a lessstable state [Ross (1995) Microbiol. Rev. 59:423-450].

Importantly, the invention demonstrates the surprising synergisticaction of AE5′ and AE3′ which together result in hFIX mRNA levels whichnot only are greater at each time point tested over the life span oftransgenic animals (FIGS. 4 B and D) as compared to hFIX mRNA levels intransgenic animals harboring vectors that lack both AE5′ and AE3′, butalso that the profile of increased human FIX mRNA levels over the lifespan of transgenic mouse recapitulates the profile of increased mouseFIX mRNA levels as a wild-type mouse ages.

Data presented herein further demonstrate that the age-relatedregulatory activity of AE5′ alone, of AE3′ alone, and of the combinationof AE5′ and AE3′ is independent of the level of expression of thetransgenes harboring them, sex, generation or zygosity status of thetransgenic animals.

The present invention is not limited to SEQ ID NOs:1 and 3 butspecifically contemplates portions thereof. As used herein the term“portion” when made in reference to a nucleic acid sequence refers to afragment of that sequence. The fragment may range in size from five (5)contiguous nucleotide residues to the entire nucleic acid sequence minusone nucleic acid residue. Thus, a nucleic acid sequence comprising “atleast a portion of” a nucleotide sequence comprises from five (5)contiguous nucleotide residues of the nucleotide sequence to the entirenucleotide sequence.

In a preferred embodiment, portions of SEQ ID NO:1 contemplated to bewithin the scope of the invention include, but are not limited to, the7-nucleotide nucleic acid sequence of the polyomavirus enhance activator3 (PEA-3) (5′-GAGGAAG-3′) (SEQ ID NO:2) which corresponds to thesequence from 2176 to 2182 of GenBank accession number K02402, and whichcorresponds to the sequence from −790 to −784 of GenBank accessionnumber K02402 when in relation to the hFIX start codon (ATG) in whichthe adenine is designated as position zero. A nucleotide sequence[5′-CAGGAAG-3′ (SEQ ID NO:40)] which is homologous to the invention'sPEA-3 nucleotide sequence was initially reported in the art as a polyomavirus enhancer, and was reported to be involved in the regulation ofexpression of various genes (e.g., collagen gene and c-fos) in severaltissues [Martin et al. (1988) Proc. Natl. Acad. Sci. 85:5839-5843; Xinet al. (1992) Genes & Develop. 6:481-496; Chotteau-Lelievre et al.(1997) Oncogene 15:937-952; Gutman and Wasylyk (1990) EMBO J.9:2241-2246]. However, the PEA-3 protein sequence [or PEA-3 relatedprotein(s)] which binds to nucleotide sequences which are homologous tothe invention's PEA-3 nucleotide sequence has not been reported to beeither liver-specific or enriched in the liver.

Other portions of SEQ ID NO: 1 included within the scope of theinvention include, for example, SEQ ID NO:33 [5′-tcgaggaagga-3′], SEQ IDNO:34 [5′-agtcgaggaaggata-3′], SEQ ID NO:35 [5′-tcagtcgaggaaggatagg-3′],SEQ ID NO:36 [5′-attcagtcgaggaaggatagggt-3′], SEQ ID NO:37[5′-ccattcagtcgaggaaggatagggtgg-3′], and SEQ ID NO:38[5′-gccattcagtcgaggaaggatagggtggta-3′], all of which include the PEA-3nucleotide sequence.

In a preferred embodiment, portions of SEQ ID NO:3 contemplated to bewithin the scope of the invention include, but are not limited to, SEQID NO:51 [5′-TTATTTTATATATATAATATATATATAAAATA-3′], SEQ ID NO:52 [5′-TATAATATA-3′], SEQ ID NO:53 [5′-CAATATAAATATATAG-3′], SEQ ID NO:54[5′-TGTGTGTGTATGCGTGTGTGTAGACACACACGCATACACACATA-3′], the combination ofSEQ ID NOs:51 and 52, i.e., SEQ ID NO:55 [5′-TTATTTTATATATATAATATATATATAAAATATATAATATA-3′], the combination of SEQ ID NOs:52and 53, i.e., SEQ ID NO:56 [5′-TATAATATACAATATAAATATATAG-3′], thecombination of SEQ ID NOs:53 and 54, i.e., SEQ ID NO:57 [5′-CAATATAAATATATAGTGTGTGTGTATGCGTGTGTGTAGACACACACGCATACACACATA-3′], the combinationof SEQ ID NOs:51, 52, 53, and 54, i.e., SEQ ID NO:58 [5′-TTATTTTATATATATAATATATATATAAAATATATAATATACAATATAAATATATAGTGTGTGTGTATGCGTGTGTGTAGACACACACGCATACACACATA-3′], the 723 bp sequence(SEQ ID NO:59) from nt 31,418 through nt 32,140 of FIG. 8, the 447 bpsequence (SEQ ID NO:60) from nt 32,244 through nt 32,690 of FIG. 8, andthe 103 bp sequence (SEQ ID NO:61) (i.e., the sl region of the 3′ UTR)from nt 32,141 through nt 32,243 of FIG. 8.

The nucleotide sequence of portions of SEQ ID NOs:1 and 3 which exhibitage-related regulatory activity may be determined using methods known inthe art, e.g., using deletion constructs (e.g., see Yang et al. (1998)J. Biol. Chem. 273:891-897). Briefly, several expression plasmids areconstructed to contain a reporter gene under the control of a promoterand of different candidate nucleotide sequences which are obtainedeither by restriction enzyme deletion of internal sequences in SEQ IDNOs:1 and 3, restriction enzyme truncation of sequences at the 5′ and/or3′ end of SEQ ID NOs:1 and 3, by the introduction of single nucleic acidbase changes by PCR into SEQ ID NOs:1 and 3, or by chemical synthesis.The gene-related regulatory activity of the different constructs isdetermined as described supra in order to determine whether thecandidate nucleotide sequence exhibits age-related regulatory activity.

The sequences of the present invention are not limited to SEQ ID NOs: 1and 3 and portions thereof, but also include homologs of SEQ ID NOs:1and 3, and homologs of portions thereof. Homologs of SEQ ID NOs:1 and 3,and of portions thereof, include, but are not limited to, nucleotidesequences having deletions, insertions or substitutions of differentnucleotides or nucleotide analogs as compared to SEQ ID NOs: 1 and 3,and of portions thereof, respectively. Such homologs may be producedusing methods well known in the art.

A “homolog” of SEQ ID NO:1 is defined as a nucleotide sequence havingmore than 63% identity and less than 100% identity with SEQ ID NO:1.Homologs of SEQ ID NO:1 are exemplified, but not limited to, SEQ IDNO:66 (5′-acccatt cagtcgagga aggatagggt ggtat-3′) which is the sequencefrom nt 2,164 to nt 2,195 of GenBank accession number kO2402, exceptthat the G at nt 1,265 is replaced with a C; SEQ ID NO:67 (5′-agccattgagtcgagga aggatagggt ggtat-3′) which is the sequence from nt 2,164 tont 2,195 of GenBank accession number kO2402, except that the C at nt2,171 is replaced with a G; SEQ ID NO:68 (5′-agccatt cagacgaggaaggatagggt ggtat-3′) is the sequence from nt 2,164 to nt 2,195 ofGenBank accession number kO2402, except that the T at nt 2,174 isreplaced with a A; SEQ ID NO:69 (5′-agccatt cagtcgagga aggatagggtggttt-3′) which is the sequence from nt 2,164 to nt 2,195 of GenBankaccession number kO2402, except that the A at nt 2,194 is replaced witha T; SEQ ID NO:70 (5′-agccatt cagtcgagga tcccaagggt ggtat-3′) which isthe sequence from nt 2,164 to nt 2,195 of GenBank accession numberkO2402, except that AGGGT beginning at nt 2,186 is replaced with TCCCA;SEQ ID NO:71 (5′-agccatt cagtcgagga aggatagggcctaat-3′) which is thesequence from nt 2,164 to nt 2,195 of GenBank accession number kO2402,except that TGGT beginnining at nt 2,190 is replaced with CCTA; SEQ IDNO:72 (5′-agaccatt cagtcgagga aggatagggt ggtat-3′) which is the sequencefrom nt 2,164 to nt 2,195 of GenBank accession number kO2402, exceptthat a A is inserted after nt 2,165; SEQ ID NO:73 (5′-agccatt cagtcgaggaaggatagcggt ggtat-3′) which is the sequence from nt 2,164 to nt 2,195 ofGenBank accession number kO2402, except that a C is inserted after nt2,187; SEQ ID NO:74 (5′-agccatt cagtcgagga aggataat-3′) which is thesequence from nt 2,164 to nt 2,195 of GenBank accession number kO2402,except that GGGTGGT beginning at nt 12,187 is deleted; and SEQ ID NO:75(5′-agccatt cgagga aggatagggt ggtat-3′) which is the sequence from nt2,164 to nt 2,195 of GenBank accession number kO2402, except that CAGTbeginning at nt 2,171 is deleted.

A “homolog” of SEQ ID NO:2 is defined as a nucleotide sequence havingmore than 75% identity and less than 100% identity with SEQ ID NO:2.Homologs of SEQ ID NO:2 include, for example, GAGGATG (SEQ ID NO:39),CAGGAAG (SEQ ID NO:40), CAGGATG (SEQ ID NO:41), GTGGAAG (SEQ ID NO:62),GTGGATG (SEQ ID NO:63), CTGGAAG (SEQ ID NO:64), CTGGATG (SEQ ID NO:65),and CAGGAAG (SEQ ID NO:84).

A “homolog” of SEQ ID NO:3 is defined as a nucleotide sequence havingless than 100% and more than 60% identity with SEQ ID NO:3. Homologs ofSEQ ID NO:3 are exemplified, but not limited to, SEQ ID NOs:76-83 shownin FIG. 13. Specifically, SEQ ID NO:76 is the sequence from nt 34,383 tont 35,655 of GenBank accession number kO2402, except that the C at nt34,390 has been replaced with a G. SEQ ID NO:77 is the sequence from nt34,383 to nt 35,655 of GenBank accession number kO2402, except that theT at nt 34,649 has been replaced with a A. SEQ ID NO:78 is the sequencefrom nt 34,383 to nt 35,655 of GenBank accession number kO2402, exceptthat the GC beginning at nt 34,959 has been replaced with a CG. SEQ IDNO:79 is the sequence from nt 34,383 to nt 35,655 of GenBank accessionnumber kO2402, except that the CATG beginning at nt 35,501 has beenreplaced with a GTAC. SEQ ID NO:80 is the sequence from nt 34,383 to nt35,655 of GenBank accession number kO2402, except that TT is insertedafter the A at nt 34,681. SEQ ID NO:81 is the sequence from nt 34,383 tont 35,655 of GenBank accession number kO2402, except that TGC isinserted after the C at nt 35,581. SEQ ID NO:82 is the sequence from nt34,383 to nt 35,655 of GenBank accession number kO2402, except that A atnt 35,636 is deleted. SEQ ID NO:83 is the sequence from nt 34,383 to nt35,655 of GenBank accession number kO2402, except that the G at nt34,383 is deleted.

A “homolog” of SEQ ID NO:59 is defined as a nucleotide sequence havingless than 100% and more than 62% identity with SEQ ID NO:59.

A “homolog” of SEQ ID NO:60 is defined as a nucleotide sequence havingless than 100% and more than 60% identity with SEQ ID NO:60.

A “homolog” of SEQ ID NO:61 is defined as a nucleotide sequence havingless than 100% and more than 60% identity with SEQ ID NO:61.

Homologs of a portion of SEQ ID NO: 1 are exemplified by homologs of thePEA-3 nucleotide sequence (SEQ ID NO:2), which include, for example,GAGGATG (SEQ ID NO:39), CAGGAAG (SEQ ID NO:40), CAGGATG (SEQ ID NO:41),GTGGAAG (SEQ ID NO:62), GTGGATG (SEQ ID NO:63), CTGGAAG (SEQ ID NO:64),CTGGATG (SEQ ID NO:65), and CAGGAAG (SEQ ID NO:84).

The present invention also contemplates functioning or functionalhomologs of SEQ ID NO:1, of portions of SEQ ID NO:1 (e.g., functionalportions of SEQ ID NOs:2, and 33-38), of SEQ ID NO:3, and of portions ofSEQ ID NO:3 (e.g., functional portions of SEQ ID NOs:51-61).

A “functional homolog” of SEQ ID NO: 1 is defined as a nucleotidesequence having more than 63% identity and less than 100% identity withSEQ ID NO:1, and which has age-related regulatory activity.Alternatively, a functional homolog of SEQ ID NO:1 is a nucleotidesequence having more than 63% identity and less than 100% identity withSEQ ID NO:1, and having liver-specific activity.

A “functional homolog” of SEQ ID NO:2 is defined as a nucleotidesequence having more than 75% identity and less than 100% identity withSEQ ID NO:2, and which has age-related regulatory activity.Alternatively, a functional homolog of SEQ ID NO:2 is a nucleotidesequence having more than 75% identity and less than 100% identity withSEQ ID NO:2, and having liver-specific activity.

A “functional homolog” of SEQ ID NO:3 is defined as a nucleotidesequence having less than 100% and more than 60% identity with SEQ IDNO:3, and which has age-related regulatory activity.

A “functional homolog” of SEQ ID NO:59 is defined as a nucleotidesequence having less than 100% and more than 62% identity with SEQ IDNO:59, and which has age-related regulatory activity.

A “functional homolog” of SEQ ID NO:60 is defined as a nucleotidesequence having less than 100% and more than 60% identity with SEQ IDNO:60, and which has age-related regulatory activity.

A “functional homolog” of SEQ ID NO:61 is defined as a nucleotidesequence having less than 100% and more than 60% identity with SEQ IDNO:61, and which has age-related regulatory activity.

The present invention is not limited to sense molecules of SEQ ID NOs: 1and 3 but contemplates within its scope antisense molecules comprising anucleic acid sequence complementary to at least a portion (e.g., aportion greater than 10 nucleotide bases in length and more preferablygreater than 100 nucleotide bases in length) of the nucleotide sequenceof SEQ ID NOs:1 and 3. These antisense molecules find use in, forexample, reducing or preventing expression of a gene (e.g. hFIX) whoseexpression is regulated by SEQ ID NOs:1 and 3.

The nucleotide sequence of SEQ ID NOs:1 and 3, portions, homologs andantisense sequences thereof may be synthesized by synthetic chemistrytechniques which are commercially available and well known in the art[see Caruthers M H et al., (1980) Nuc. Acids Res. Symp. Ser. 215-223;Horn T. et al., (1980) Nuc. Acids Res. Symp. Ser. 225-232].Additionally, fragments of SEQ ID NOs:1 and 3 can be made by treatmentof SEQ ID NOs:1 and 3 with restriction enzymes followed by purificationof the fragments by gel electrophoresis. Alternatively, sequences mayalso be produced using the polymerase chain reaction (PCR) as describedby Mullis [U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, all ofwhich are hereby incorporated by reference]. SEQ ID NOs:1 and 3,portions, homologs and antisense sequences thereof may be ligated toeach other or to heterologous nucleic acid sequences using methods wellknown in the art.

The nucleotide sequence of synthesized sequences may be confirmed usingcommercially available kits as well as using methods well known in theart which utilize enzymes such as the Klenow fragment of DNA polymeraseI, Sequenase®, Taq DNA polymerase, or thermostable T7 polymerase.Capillary electrophoresis may also be used to analyze the size andconfirm the nucleotide sequence of the products of nucleic acidsynthesis, restriction enzyme digestion or PCR amplification.

It is readily appreciated by those in the art that the sequences of thepresent invention may be used in a variety of ways. For example, thenucleic acid sequences of the invention and portions thereof can be usedas probes for the detection and isolation of functional homologs of AE5′and AE3′, amplification of homologous nucleotide sequences, age-relatedand/or liver-specific expression of a nucleotide sequence of interest inan animal, gene therapy, and reducing factor IX levels in an animal.

B. Using Probes to Identify and Isolate Homologs of AE5′ and AE3′

The invention provided herein is not limited to SEQ ID NO:1 and 3,homologs and portions thereof having age-related regulatory activity,but includes sequences having no age-related regulatory activity (i.e.,non-functional homologs and non-functional portions of homologs). Theuse of such sequences may be desirable, for example, where a portion ofSEQ ID NOs:1 and 3 is used as a probe to detect the presence of SEQ IDNOs:1 and 3, respectively, or of portions thereof in a sample.

As used herein, the term “probe” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, recombinantly or by PCR amplification, which is capableof hybridizing to a nucleotide sequence of interest. A probe may besingle-stranded or double-stranded. It is contemplated that any probeused in the present invention will be labelled with any “reportermolecule,” so that it is detectable in any detection system including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, calorimetric,gravimetric, magnetic, and luminescent systems. It is not intended thatthe present invention be limited to any particular detection system orlabel.

The probes provided herein are useful in the detection, identificationand isolation of, for example, sequences such as those listed as SEQ IDNOs:1 and 3 as well as of homologs thereof. Preferred probes are ofsufficient length (e.g., from about 9 nucleotides to about 20nucleotides or more in length) such that high stringency hybridizationmay be employed. In one embodiment, probes from 20 to 50 nucleotidebases in length are employed.

C. Using Primers to Amplify at Least a Portion of AE5′ and AE3′

The invention provided herein is not limited to SEQ ID NOs:1 and 3,homologs and portions thereof having age-related regulatory activity,but includes sequences having no age-related regulatory activity. Thismay be desirable, for example, where a portion of the nucleic acidsequences set forth as SEQ ID NOs:1 and 3 is used as a primer for theamplification of nucleic acid sequences by, for example, polymerasechain reactions (PCR) or reverse transcription-polymerase chainreactions (RT-PCR). The term “amplification” is defined as theproduction of additional copies of a nucleic acid sequence and isgenerally carried out using polymerase chain reaction technologies wellknown in the art [Dieffenbach C W and G S Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview N.Y.]. As usedherein, the term “polymerase chain reaction” (“PCR”) refers to themethod of K. B. Mullis disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202and 4,965,188, all of which are hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare the to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;and/or incorporation of ³²P-labeled deoxyribonucleotide triphosphates,such as dCTP or dATP, into the amplified segment). In addition togenomic DNA, any nucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications. Amplified target sequencesmay be used to obtain segments of DNA (e.g., genes) for the constructionof targeting vectors, transgenes, etc.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long (e.g.,from about 9 nucleotides to about 20 nucleotides or more in length) toprime the synthesis of extension products in the presence of theinducing agent. Suitable lengths of the primers may be empiricallydetermined and depend on factors such as temperature, source of primerand the use of the method. In one embodiment, the present inventionemploys primers from 20 to 50 nucleotide bases in length.

The primers contemplated by the invention are useful in, for example,identifying sequences which are homologous to AE5′ and AE3′ in mammals,yeast, bacteria, and in other organisms.

D. Methods for Regulating Gene Expression

The present invention provides methods for regulating expression of anucleotide sequence of interest over a period of time in a cell ormulticellular organism. Specifically, gene expression is preferablyregulated in a multicellular organism. In one embodiment, expression ofa nucleotide sequence of interest is stabilized such that the level ofmRNA and/or protein encoded by the nucleotide sequence of interestremains relatively unchanged at different times during the life of theorganism. In an alternative embodiment, expression of a nucleotidesequence of interest is increased. Increased expression means that thelevel of mRNA and/or protein encoded by the nucleotide sequence ofinterest at a given time point is greater than the level of mRNA and/orprotein, respectively, at an earlier time point during the life of theorganism or cell. Alternatively, increased expression means that thelevel of mRNA and/or protein encoded by the nucleotide sequence ofinterest is greater than the level of mRNA and/or protein, respectively,at the same time point in the life of the organism or cell as comparedto the level of mRNA and/or protein when expressed in the absence of thesequences of the invention.

In one embodiment, regulating expression of a nucleotide sequence ofinterest over a period of time is accomplished by introducing into ahost cell a vector that contains a nucleotide sequence of interestoperably linked to a promoter sequence and to sequences provided hereinwhich have age-related regulatory activity. The transfected host cell isallowed to develop into a transgenic animal in which the nucleotidesequence of interest is expressed in at least one tissue. These stepsare further described below for specific embodiments.

1. Expression Constructs

In one embodiment of the methods of the invention for regulatingexpression of a nucleotide sequence of interest in an age-related mannerand/or to liver tissue, a vector is constructed in which the nucleicacid sequences of the invention (e.g., AE5′ alone, AE3′ alone, or acombination of AE5′ and AE3′) are operably linked to a promoter sequenceand to a nucleotide sequence of interest. In one embodiment, thenucleotide sequence of interest is the coding region of the hFIX gene(Example 1). In another embodiment the nucleotide sequence of interestis the coding region of the protein C gene (Example 7).

The invention is not limited to coding sequences of the hFIX gene orprotein C gene. Rather, any nucleotide acid sequence whose expression isdesired to be regulated by sequences provided herein are contemplated tobe within the scope of this invention. Such nucleotide sequencesinclude, but are not limited to, coding sequences of structural geneswhich encode a protein [e.g., reporter genes, selection marker genes,oncogenes, drug resistance genes, growth factor genes, activator protein1 gene, activator protein 2 gene, Sp1 gene, etc.]. In one preferredembodiment, the structural gene is the human α1-antitrypsin gene (FIG.10) (SEQ ID NO:42) which encodes a plasma proteinase inhibitor used fortreating emphysema. In another preferred embodiment, the structural geneis one encoding the human antithrombin III (FIG. 11) (SEQ ID NO:43)which is a plasma proteinase inhibitor for activated blood coagulationfactors and whose activity is increased by heparin. In yet anotherpreferred embodiment, the structural gene is the gene encoding the PEA-3protein (FIG. 9) (SEQ ID NO:47) and/or its related protein, which hasbeen shown to bind specifically to homologs of the PEA-3 nucleotidesequence (SEQ ID NO:2) disclosed herein.

The invention is not limited to using a single nucleotide sequence ofinterest in operable combination with the sequences of the invention.Rather, a plurality (i.e., more than one) of nucleotide sequences ofinterest may be ligated in tandem such that their expression isregulated by the regulatory sequences of the invention. A plurality ofcoding sequences may be desirable, for example, where it is useful toexpress a transcription product of more than one gene to permitinteraction of these transcriptional products. Alternatively, aplurality of coding sequences may be desirable where one of the genesequences is a reporter gene sequence. For example, it may beadvantageous to place a coding sequence of a reporter gene in tandemwith the coding sequence of a gene of interest such that expression ofthe coding region of both the reporter gene and the gene of interest isregulated by the sequences of the invention. Expression of the reportergene usually correlates with expression of the gene of interest.Examples of reporter gene sequences include the sequences encoding theenzymes β-galactosidase and luciferase. Fusion genes may also bedesirable to facilitate purification of the expressed protein. Forexample, the heterologous sequence which encodes protein A allowspurification of the fusion protein on immobilized immunoglobulin. Otheraffinity traps are well known in the art and can be utilized toadvantage in purifying the expressed fusion protein. For example, pGEXvectors (Promega, Madison Wis.) may be used to express the polypeptidesof interest as a fusion protein with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Other fusion polypeptidesuseful in the purification of proteins of interest are commerciallyavailable, including histidine tails (which bind to Ni²⁺), biotin (whichbinds to streptavidin), and maltose-binding protein (MBP) (which bindsto amylose). Proteins made in such systems may be designed to includeheparin, thrombin or factor XA protease cleavage sites so that thecloned polypeptide of interest can be released at will from theheterologous polypeptide moiety to which it is fused.

One of skill in the art would understand that where a plurality ofnucleotide sequences of interest is operably linked to sequences of thepresent invention, the nucleotide sequences of interest may be eithercontiguous or separated by intervening polynucleotide sequences, so longas the nucleic acid sequences of interest are operably linked to thepromoter sequence, and so long as the sequences of the invention areoperably linked to the promoter sequence.

While specific preferred embodiments used herein disclose the use of thehFIX promoter and the CMV promoter, it is not intended that theinvention be limited to the type or source of the promoter sequencewhich is operably linked to the sequences of the invention. Any promoterwhose activity is desired to be regulated by the sequences providedherein is contemplated to be within the scope of the invention.Exemplary promoters include the tRNA promoter, 5S rRNA promoters,histone gene promoters, RSV promoter (can be isolated from vectorplasmid pRc/RSV from Invitrogen), retrovirus LTR promoter (can beisolated from vector plasmid pLXSN from Clontech) SV40 promoter (locatedbetween positions +3530 to +3192 in vector plasmid pCR3 fromInvitrogen), PEPCK promoter, MT promoter, SRα promoter, P450 familypromoters, GAL7 promoter, T₇ promoter having the 23-bp sequence (SEQ IDNO:44) 5′-TAATACGACTCACTATAGGGCGA-3′, T₃ promoter having the 24-bpsequence (SEQ ID NO:45) 5′-TTATTAACCCTCACTAAAGGGAAG-3′, SP6 promoterhaving the 23-bp sequence (SEQ ID NO:46) 5′-ATTTAGGTGACACTATAGAATAC-3′,and K11 promoter. The T₇ promoter, T₃ promoter, SP6 promoter andK1]promoter have been described in U.S. Pat. No. 5,591,601, the entirecontents of which are incorporated by reference.

Nor is the invention intended to be limited to the use of a singlepromoter. For example, chimeric promoters (i.e., two or more promoterswhich are derived from at least one gene) are expressly contemplated tobe within the scope of the invention. Such chimeric promoters may bedesirable where, for example, chimeric promoters result in increasedlevels of expression of an operably linked downstream coding sequence.Chimeric promoters are known in the art and include, for example, thedouble Tet promoter [Kistner et al. (1996) Proc. Natl. Acad. Sci. USA93:10933-10938], and the U1 snRNA promoter-CMV promoter/enhancer[Bartlett et al. (1996) Proc. Natl. Acad. Sci. USA 93:8852-8857].

Expression vectors in which expression of a nucleic acid sequence ofinterest is regulated by sequences of the invention may be constructedusing the teachings of the present invention in conjunction withtechniques well known in the art. [Sambrook et al. (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.;Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley& Sons, New York N.Y.]. Briefly, the nucleic acid sequence of interestis placed in operable combination with a promoter sequence and sequencesof the invention in the presence of transcription and translationregulatory sequences, including initiation signals such as a start codon(i.e., ATG), enhancers, and transcription termination signals. The ATGinitiation codon must be in the correct reading frame to ensuretranslation of the entire heterologous nucleotide sequence.Transcription termination signals are placed downstream of theheterologous nucleic acid sequence and include polyadenylation sequenceswhich are exemplified by, but not limited to, SV40 poly-A sequence, hINVpoly-A sequence, or bovine growth hormone poly-A sequence, etc.

Other regulatory sequences which may affect RNA stability as well asenhancers (i.e., a sequence which when activated results in an increasein the basal rate of transcription of a gene) and silencers (i.e., asequence involved in reducing expression of a gene) may also beincluded. These regulatory sequences may be relativelyposition-insensitive, i.e., the regulatory element will functioncorrectly even if positioned differently in relation to the heterologousnucleotide sequence in the construct as compared to its position inrelation to the corresponding heterologous nucleotide sequence in thegenome. For example, an enhancer may be located at different distancesfrom the promoter sequence, in a different orientation, and/or in adifferent linear order. Thus, an enhancer that is located 3′ to apromoter sequence in germline configuration might be located 5′ to thepromoter sequence in the construct.

2. Host Cells

Host cells are transformed with expression vectors which contain thesequences of the invention in operable combination with a nucleic acidsequence of interest using methods known in the art. The term“transformation” as used herein refers to the introduction of atransgene into a cell. The term “transgene” as used herein refers to anynucleic acid sequence which is introduced into the genome of a cell byexperimental manipulations.

The term “transgene” as used herein refers to any nucleic acid sequencewhich is introduced into the genome of a cell by experimentalmanipulations. A transgene may be an “endogenous DNA sequence,” or a“heterologous DNA sequence.” The term “endogenous DNA sequence” refersto a nucleotide sequence which is naturally found in the cell into whichit is introduced so long as it does not contain some modification (e.g.,a point mutation, the presence of a selectable marker gene, etc.)relative to the naturally-occurring sequence. The terms “heterologousDNA sequence” and “foreign DNA sequence” refer to a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA is notendogenous to the cell into which it is introduced, but has beenobtained from another cell. Heterologous DNA also includes an endogenousDNA sequence which contains some modification (e.g., a point mutation,the presence of a selectable marker gene, etc.) relative to thenaturally-occurring gene. Generally, although not necessarily,heterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

Transformation may be accomplished by a variety of means known to theart including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, biolistics (i.e., particlebombardment) and the like.

Transformation of a cell may be stable or transient. The term “transienttransformation” or “transiently transformed” refers to the introductionof one or more transgenes into a cell in the absence of integration ofthe transgene into the host cell's genome. Transient transformation maybe detected by, for example, enzyme-linked immunosorbent assay (ELISA)which detects the presence of a polypeptide encoded by one or more ofthe transgenes. Alternatively, transient transformation may be detectedby detecting the activity of the protein encoded by the transgene. Forexample, the activity of β-glucuronidase (GUS) which is encoded by theuid A gene may be detected using either a histochemical assay of GUSenzyme activity by staining with X-gluc which gives a blue precipitatein the presence of the GUS enzyme, or a chemiluminescent assay using theGUS-Light kit (Tropix). The term “transient transformant” refers to acell which has transiently incorporated one or more transgenes. Incontrast, the term “stable transformation” or “stably transformed”refers to the introduction and integration of one or more transgenesinto the genome of a cell. Stable transformation of a cell may bedetected by Southern blot hybridization of genomic DNA of the cell withnucleic acid sequences which are capable of binding to one or more ofthe transgenes. Alternatively, stable transformation of a cell may alsobe detected by the polymerase chain reaction (PCR) of genomic DNA of thecell to amplify transgene sequences. The term “stable transformant”refers to a cell which has stably integrated one or more transgenes intothe genomic DNA. Thus, a stable transformant is distinguished from atransient transformant in that, whereas genomic DNA from the stabletransformant contains one or more transgenes, genomic DNA from thetransient transformant does not contain a transgene.

Suitable host cells include bacterial, yeast, plant, insect, andmammalian cells. In one embodiment the host cell is mammalian. In apreferred embodiment, the mammalian host cell is a mouse fertilized eggcell. In an alternative embodiment, the mammalian host cell is a HepG2cell (ATCC number HB8065), a fibroblast cell (e.g., ATCC number CCL110), a myoblast cell (e.g., Clonetics, catalog # SkMC), and anendothelial cell (e.g., human umbilical cord endothelial cells; ATCCnumber CRL 1730).

In one embodiment, the host cell is transformed both with an expressionvector which contains the sequences of the invention in operablecombination with the nucleic acid sequences of interest, as well as withan expression vector which expresses the PEA-3 protein (Example 6). Suchco-transformation may be desirable, for example, where expression of thenucleotide sequence of interest is regulated by AE5′ or portions orhomologs thereof which contain homologs of the PEA-3 nucleotide sequenceto which the PEA-3 protein binds. In one embodiment, expression of thePEA-3 protein is under the control of the LTR promoter of the Moloneymurine leukemia virus (MOLV) which is capable of driving expression ofoperably linked genes in several cell types. Transient expression assaysare suitable for determining the relative promoter activities inexpressing desirable PEA-3 protein levels.

Any number of selection systems may be used to recover transfectedcells. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler M et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy I et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate [Wigler M et al., (1980) Proc Natl Acad Sci 77:3567-70];npt, which confers resistance to the aminoglycosides neomycin and G-418[Colbere-Garapin F et al., (1981) J. Mol. Biol. 150:1-14] and als orpat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine [Hartman S C and R C Mulligan(1988) Proc Natl Acad Sci 85:8047-51]. Recently, the use of a reportergene system which expresses visible markers has gained popularity withsuch markers as β-glucuronidase and its substrate (GUS), luciferase andits substrate (luciferin), and β-galactosidase and its substrate (X-Gal)being widely used not only to identify transformants, but also toquantify the amount of transient or stable protein expressionattributable to a specific vector system [Rhodes C A et al. (1995)Methods Mol Biol 55:121-131].

The presence or expression of the reporter gene usually indicates thepresence or expression, respectively, of the tandem heterologous nucleicacid sequence as well. However, it is preferred that the presence andexpression of the desired heterologous nucleic acid sequence beconfirmed. This is accomplished by procedures known in the art whichinclude DNA-DNA or DNA-RNA hybridization or amplification using probes,or fragments of the heterologous nucleic acid sequence. For example,Fluorescent In Situ Hybridization (FISH) can be used to detect theheterologous nucleic acid sequence in cells. Several guides to FISHtechniques are available, e.g., Gall et al. Meth. Enzymol. 21:470-480(1981); Angerer et al., in “Genetic Engineering: Principles andMethods,” Setlow & Hollaender, Eds. Vol. 7 pp. 43-65, Plenum Press, NewYork (1985). Alternatively, DNA or RNA can be isolated from cells fordetection of the transgene by Southern or Northern hybridization or byamplification based assays. Nucleic acid amplification based assaysinvolve the use of oligonucleotides or oligomers based on the nucleotidesequence of interest in order to detect cells and tissues which containthe DNA or RNA encoding the transgene of interest. As used herein, theterms “oligonucleotides” and “oligomers” refer to a nucleic acidsequence of at least about five (5) contiguous nucleotide residues andas many as about sixty (60) nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20-25 nucleotides, which can beused as a probe or amplimer. Standard PCR methods useful in the presentinvention are described by Innis et al. (Eds.), “PCR Protocols: A Guideto Methods and Applications,” Academic Press, San Diego (1990).

Yet another alternative for the detection of heterologous nucleic acidsequences includes detecting the polypeptide product of transcription ofthe heterologous nucleotide sequence. A variety of protocols whichemploy polyclonal or monoclonal antibodies specific for the proteinproduct are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A competitive binding assay may also beused. Alternatively, a two-site, monoclonal-based immunoassay whichutilizes monoclonal antibodies that are reactive to two non-interferingepitopes on the protein of interest may be employed. These and otherassays are described in, among other places, Hampton R et al. (1990),Serological Methods a Laboratory Manual, APS Press, St Paul Minn.), andMaddox D E et al. (1983), J. Exp. Med. 158:1211.

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting related sequences include oligolabeling, nick translation,end-labeling or PCR amplification using a labeled nucleotide.Alternatively, the nucleotide sequence of interest, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A numberof companies such as Pharmacia Biotech (Piscataway N.J.), Promega(Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like.

Host cells transformed with expression vectors containing the sequencesprovided herein are useful for age-related expression of recombinantproteins of interest. Host cells transformed with expression vectorscontaining the invention's sequences may be part of a tissue or organ ofa living animal. A “living animal” as used herein refers to anymulticellular animal (e.g., humans, non-human primates, ovines, bovines,ruminants, lagomorphs, porcines, caprines, equines, canines, felines,aves, etc.) into whose cells the sequences provided herein may beintroduced. Where the host cells (e.g., fertilized egg cells) arecapable of generating a multicellular organism, these cells whentransformed with expression vectors containing the sequences of theinvention are useful in generating transgenic animals which exhibitage-related and/or liver-specific expression of nucleotide sequences ofinterest.

3. Transgenic Animals

The present invention provides transgenic non-human animals whichexpress a nucleotide sequence of interest in an age-related manner.These animals provide useful models for diseases (e.g., thrombosis,cardiovascular diseases, diabetes, Alzheimer's disease, cancer,osteoporosis, osteoarthritis, Parkinson's disease, dementia) which areassociated with increasing age, as well a for screening candidatetherapeutic agents against such diseases. These transgenic animals arealso useful in studies of normal phenomena, such as ageing, generegulation, etc. In one embodiment, the invention discloses transgenicmice which express in an age-related manner the exemplary hFIX codingsequence under the control of AE5′ and/or AE3′ (Example 3).

The term “age-related manner” when made in reference to the expressionof a nucleotide sequence of interest is a relative term which refers toan increase over a period of time in the quantity of mRNA and/or proteinencoded by the nucleotide sequence of interest when the nucleotidesequence of interest is operably linked to a promoter and to a nucleicacid sequence which has age-related regulatory activity, as compared tothe quantity of mRNA and/or protein, respectively, encoded by thenucleotide sequence of interest when the nucleotide sequence of interestis operably linked to the promoter in the absence of the nucleic acidsequence which has age-related regulatory activity. Thus, the term“age-related” when made in reference to expression of a nucleotidesequence of interest by a transgenic animal means that the transgenicanimal expresses the nucleotide sequence of interest in an age-relatedmanner.

For example, in one embodiment, the invention demonstrates that hFIX isexpressed in an age-related manner in transgenic mice which harbor atransgene (−416FIXm1/1.4) (FIG. 2B) which contains hFIX under thecontrol of the hFIX promoter and the regulatory control of AE3′ ascompared to expression of hFIX in transgenic mice which harbor atransgene (−416FIXm1) (FIG. 2A) in which hFIX is under the control ofthe hFIX promoter in the absence of AE3′. While transgenic miceharboring the −416FIXm1/1.4 construct showed decreasing hFIX activitylevels over a period of time (e.g., from 1 to 9 months of age), thisdecrease was less than the decrease in hFIX activity levels which wasobserved in transgenic mice harboring the −416FIXm1 construct over thesame period of time.

In another embodiment, the invention discloses that hFIX is expressed inan age-related manner in transgenic mice which harbor transgenes(−802FIXm1, −2231FIXm1, and −416FIXm1/AE5′) (FIGS. 4A, C and E) each ofwhich contains hFIX under the control of the hFIX promoter and theregulatory control of AE5′ as compared to expression of hFIX intransgenic mice which harbor a transgenes (−416FIXm1 and −770FIXm1)(FIGS. 2A and E) in which hFIX is under the control of the hFIX promoterin the absence of AE5′. Transgenic mice harboring each of the −802FIXm1,−2231FIXm1, and −416FIXm1/AE5′ constructs showed relatively unchangedhFIX activity levels over a period of time (e.g., from 1 to 7 months ofage) while transgenic mice harboring either the −416FIXm1 or −770FIXm1construct showed decreasing hFIX activity levels over the same timeperiod.

In an additional embodiment, the invention shows that hFIX is expressedin an age-related manner in transgenic mice which harbor transgenes(−802FIXm1/1.4 and −2231FIXm1/1.4) (FIGS. 4B and D) each of whichcontains hFIX under the control of the hFIX promoter and the regulatorycontrol of both AE3′ and AE5′ as compared to expression of hFIX intransgenic mice which harbor a transgene (−770FIXm1) (FIG. 2E) in whichhFIX is under the control of the hFIX promoter in the absence of bothAE3′ and AE5′. Transgenic mice harboring either the −802FIXm1/1.4 or the−2231FIXm1/1.4 construct showed increasing levels of hFIX activity overa period of time (e.g., 1 to 3 months of age) as compared to decreasinghFIX activity levels over the same period of time in transgenic miceharboring the −770FIXm1 construct.

The present invention also provides transgenic non-human animals whichexpress a nucleotide sequence of interest in a liver-specific manner.These animals are useful for targeting expression of a nucleotidesequence of interest to the liver. Examples of nucleotide sequences ofinterest are those which encode blood coagulation factors (e.g., factorVIII, factor VII, factor X and prothrombin) whose deficiency is known toplay a role in abnormal bleeding disorders. Other examples of nucleotidesequences of interest include those which encode blood coagulationregulators and/or inhibitors (e.g., protein C, antithrombin III, andtissue factor pathway inhibitor [TFPI]) whose deficiency results inthrombosis, α1-antitrypsin whose deficiency results in emhysima, andLDL-receptor whose deficiency results in hypercholestrolemia.

Yet other examples of a nucleotide sequence of interest include thoseencoding enzymes involved in specific metabolic defects (e.g., ureacycle enzymes, especially ornithine transcarbamylase, argininosuccinatesynthase, and carbamyl phosphate synthase); receptors (e.g., LDLreceptor); toxins; thymidine kinase to ablate specific cells or tissues;ion channels (e.g., chloride channel of cystic fibrosis); membranetransporters (e.g., glucose transporter); and cytoskeletal proteins(e.g., dystrophin). The nucleotide sequence of interest may be ofsynthetic, cDNA, or genomic origin, or a combination thereof. Thenucleotide sequence of interest may be one which occurs in nature, anon-naturally occurring gene which nonetheless encodes a naturallyoccurring polypeptide, or a gene which encodes a recognizable mutant ofsuch a polypeptide. It may also encode an mRNA which will be “antisense”to a DNA found or to an mRNA normally transcribed in the host cell, butwhich antisense RNA is not itself translatable into a protein. In oneembodiment, the invention discloses transgenic mice which express in aliver-specific manner the exemplary hFIX coding sequence under thecontrol of AE5′ (Example 3).

The term “liver-specific manner” as used herein in reference to theexpression of a nucleotide sequence of interest in a transgenic animalis a relative term which means that the quantity of mRNA and/or proteinencoded in liver tissue by the nucleotide sequence of interest isgreater than, preferably two times greater, more preferably five timesgreater, and most preferably ten times greater, than the quantity ofmRNA and/or protein encoded by the nucleotide sequence of interest intissues other than liver tissue of the same transgenic animal asdetected by Northern blot hybridization and/or by the activity of theencoded protein as described herein. Thus, the term “liver-specific”when made in reference to expression of a nucleotide sequence ofinterest by a transgenic animal means that the transgenic animalexpresses the nucleotide sequence of interest in an liver-specificmanner.

A first step in the generation of the transgenic animals of theinvention is the introduction of a construct containing nucleic acidsequences of interest under the expression regulatory control ofsequences of the invention into target cells. Several methods areavailable for accomplishing this, including microinjection, retroviralinfection, and implantation of embryonic stem cells. These methods arediscussed as follows.

i. Microinjection Methods

Direct microinjection of expression vectors into pronuclei of fertilizedeggs is the preferred, and most prevalent, technique for introducingheterologous nucleic acid sequences into the germ line. Technicalaspects of the microinjection procedure and important parameters foroptimizing integration of nucleic acid sequences have been previouslydescribed [Hogan et al., (1986) Manipulation of the Mouse Embryo: ALaboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Lab.].

Once the expression vector has been injected into the fertilized eggcell, the cell is implanted into the uterus of a pseudopregnant femaleand allowed to develop into an animal. Of the founder transgenic animalsborn, 70% carry the expression vector sequence in all of their cells,including the germ cells. The remaining 30% of the transgenic animalsare chimeric in somatic and germ cells because integration of theexpression vector sequence occurs after one or more rounds ofreplication. Heterozygous and homozygous animals can then be produced byinterbreeding founder transgenics. This method has been successful inproducing transgenic mice, sheep, pigs, rabbits and cattle [Hammer etal., (1986) J. Animal Sci.:63:269; Hammer et al., (1985) Nature315:680-683].

ii. Retroviral Methods

Retroviral infection of pre-implantation embryos with geneticallyengineered retroviruses may also be used to introduce transgenes into ananimal cell. For example, blastomeres have been used as targets forretroviral infection [Jaenisch, (1976) Proc. Natl. Acad. Sci USA73:1260-1264]. Transfection is typically achieved using areplication-defective retrovirus carrying the transgene [Jahner et al.,(1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten et al.,(1985) Proc. Natl. Acad Sci USA 82:6148-6152]. Transfection is obtained,for example, by culturing eight-cell embryos, from which the zonapellucida has been removed with fibroblasts which produce the virus [Vander Putten (1985), supra; Stewart et al., (1987) EMBO J. 6:383-388]. Thetransfected embryos are then transferred to foster mothers for continueddevelopment. Alternatively, infection can be performed at a later stage.Virus or virus-producing cells can be injected into the blastocoele[Jahner et al., (1982) Nature 298:623-628]. Yet another alternativemethod involves intrauterine retroviral infection of the midgestationembryos [Jahner et al. (1982), supra].

The advantages of retroviral infection methods include the ease oftransfection and the insertion of a single copy of the transgene, whichis flanked by the retroviral long terminal repeats (LTRs), into thechromosome. However, this method is not a preferred method because mostof the founders will show mosaicism since infection occurs after celldivision has begun. This necessitates outbreeding to establishhomozygous and heterozygous lines suitable for analysis of geneexpression. More importantly, the retroviral LTR sequences may interferewith the activity of the hINV upstream sequences in directing expressionof the heterologous nucleic aid sequences.

iii. Embryonic Stem Cell Implantation

Another method of introducing transgenes into the germ line involvesusing embryonic stem (ES) cells as recipients of the expression vector.ES cells are pluripotent cells directly derived from the inner cell massof blastocysts [Doetchman et al., (1988) Dev. Biol. 127:224-227], frominner cell masses [Tokunaga et al., (1989) Jpn. J. Anim. Reprod.35:173-178], from disaggregated morulae [Eistetter, (1989) Dev. Gro.Differ. 31:275-282] or from primordial germ cells [Matsui et al., (1992)Cell 70:841-847]. Expression vectors can be introduced into ES cellsusing any method which is suitable for gene transfer into cells, e.g.,by transfection, cell fusion, electroporation, microinjection, DNAviruses, and RNA viruses [Johnson et al., (1989) Fetal Ther. 4 (Suppl.1):28-39].

The advantages of using ES cells include their ability to form permanentcell lines in vitro, thus providing an unlimited source of geneticmaterial. Additionally ES cells are the most pluripotent cultured animalcells known. For example, when ES cells are injected into an intactblastocyst cavity or under the zona pellucida, at the morula stageembryo, ES cells are capable of contributing to all somatic tissuesincluding the germ line in the resulting chimeras.

Once the expression vector has been introduced into an ES cell, themodified ES cell is then introduced back into the embryonic environmentfor expression and subsequent transmission to progeny animals. The mostcommonly used method is the injection of several ES cells into theblastocoel cavity of intact blastocysts [Bradley et al., (1984) Nature309:225-256]. Alternatively, a clump of ES cells may be sandwichedbetween two eight-cell embryos [Bradley et al., (1987) in“Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,” Ed.Robertson E. J. (IRL, Oxford, U.K.), pp. 113-151; Nagy et al., (1990)Development 110:815-821]. Both methods result in germ line transmissionat high frequency.

Target cells which contain the heterologous nucleic acid sequences arerecovered, and the presence of the heterologous nucleic acid sequence inthe target cells as well as in the animal is accomplished as describedsupra.

E. Gene Therapy

The regulatory nucleic acid sequences provided herein may be used forgene therapy applications in both non-human animals as well as inhumans. For example, the regulatory nucleic acid sequences of theinvention may be introduced into cells using an expression vector whichencodes a polypeptide sequence of interest using a variety of meansknown in the art to be useful both for delivery in vivo and ex vivo,including (1) recombinant retroviral transduction, (2) recombinantadenoviral vectors, (3) targeted cationic liposomes, and (4) genetransfer using biolistics, as described in the following sections.

1. Recombinant Retroviral Transduction

Retroviral vectors encoding polypeptides of interest may be used for theexpression of the polypeptides in any desired cell, such as primarytumor cells. The transfer of polypeptides of interest using retrovirusesmay be made more efficient by increasing the titer of the virus encodingthe polypeptides of interest and increasing the transduction efficiency.To increase the virus titer, the retroviral construct may be designed toinclude a selectable marker (e.g., neo gene), and cells harboring theretroviral construct are selected by growth in the presence of asuitable selective agent (e.g., G418) followed by expansion of clonesproducing the highest titers of virus. To improve the transductionefficiency, retrovirus are used in combination with liposomes orpoly-L-ornithine or polylysine to enhance virus uptake.

Another way to improve gene transfer efficiency using retroviruses is toincrease the targeting efficiency. Many tumor cells includingglioblastomas and melanomas express excess levels of the transferrinreceptor. Transferrin has been used to increase the transductionefficiency of adenovirus in combination with polylysine. Several recentreports demonstrated that replacing the SU (surface) domain of the envgene of a retrovirus can increase receptor-mediated transductionefficiency. The human transferrin gene is 2097 bp long and its insertioninto the SU domain of the env gene of MLV vector may not produce astable Env product. However, since earlier studies have suggested thatthe modified Env fusion protein requires the native Env for stableassembly and efficient entry, co-transfection of the transferrin-envfusion gene with the native env gene may be used to produce retrovirusparticles bearing a mixture of wild type and recombinant Env. The genetransfer efficiency of the new vector may be examined by transducingtumor cells expressing high levels of transferrin receptor.

2. Recombinant Adenoviral Vectors

Recombinant adenoviruses can accommodate relatively large segments offoreign DNA (˜7 kb), and have the advantage of a broad host cell rangeand high titer virus production. Adenoviruses have been used in vivo inrats to efficiently deliver genes to the liver and the pancreatic islets[reviewed in Becker et al. (1994) In Protein Expression in Animal Cells,Roth et al. eds.] and to the central nervous system [Davidson et al.(1993) Nature Genet. 3:219]. Rat livers have also been efficientlytransduced ex vivo and then re-implanted [Shaked et al. (1994)Transplantation 57:1508]. Thus, the present invention contemplates exvivo transfection followed by transplantation of the transfected cellsor organ.

The replication defective recombinant adenoviruses are preferablyemployed; these viruses contain a deletion of the key immediate earlygenes E1a and E1b. To generate and propagate recombinant viruses, apackaging cell line such as 293 cells which supply the E1a and E2aproteins in trans is employed. Recombinant adenoviruses are created bymaking use of intracellular recombination between a much larger plasmidencoding most of the viral genome and a small plasmid containing thenucleotide sequence of interest flanked by regions of homology with theviral integration site. Standard methods may be used to construct therecombinant adenoviruses [Graham and Prevec (1991) Meth. Mol. Biol.7:109-128; Becker et al. (1994) In Protein Expression in Animal Cells,Roth et al. eds.]. Briefly, each plasmid is co-transfected together withpJM17 (Microbix Systems, Toronto) into sub-confluent monolayers of 293cells (ATCC CRL 1573) using calcium phosphate precipitation and aglycerol shock. Initial recombinant viral stocks are titered onmonolayers of 293 cells, and isolated single plaques are obtained andtested for expression of the polypeptide of interest using ELISA. Viralstocks are amplified and titered on 293 cells, and stored in aliquots at−70° C.; if necessary, stocks are concentrated by centrifugation ondensity gradients. To infect tumor cells with recombinant adenoviruses,freshly isolated tumor cells are mixed with adenoviral stocks in aminimal volume. Titers of stocks are typically 10⁵-10⁸/ml. Medium isreplaced after several hours and the cells are followed for expressionof the recombinant adenoviral-encoded polypeptide of interest (e.g.,reporter genes).

A potential drawback of using an adenoviral delivery system is that thetransduced cells may retain or express small quantities of adenoviralantigens on their surface. “Second generation” adenoviral vectors whichcontain deletions in the E2a gene are available and are associated withless inflammation in the recipient and a longer period of expression ofthe gene of interest [Engelhardt et al. (1994) Proc. Natl. Acad. Sci.USA 91:6196]. If necessary, nucleic acid sequences encoding polypeptidesof interest are inserted into second generation adenoviral vectors.

Recently, adenoassociated virus (AAV) vectors and chimeric lentivirusvectors have also been shown promise in the expression of polypeptidesequences of interest.

3. Targeted Cationic Liposomes

Cationic liposomes have proven to be a safe and effective means forinducing the transient expression of DNA in target cells [Ledley (1995)Human Gene Ther. 6:1129]. Clinical trials are underway using cationicliposomes to introduce the CFTR gene into the lungs of cystic fibrosispatients [Caplen et al. (1994) Gene Ther. 1:139 and Alton et al. (1993)Nature Genet. 5:135] or to introduce, by direct intra-tumor injection,the T cell costimulator B7-1 into malignant melanoma lesions in order toinduce a cell-mediated immune response [Nabel et al. (1993) Proc. Natl.Acad. Sci. USA 90:11307].

Cationic liposomes (e.g., DOTAP/DOPE) and ligand-targeted cationicliposomes may be employed for the delivery of polypeptides of interestto tumor cells. Recently, in addition to cationic liposomes, neutralliposomes have also been reproted to also be useful in targeing ligandsto cells. Ligand-targeted liposomes are made by covalently attachingligands or antibodies to the surface of the cationic liposome. Forexample, when glioblastoma cells are to be targeted, transferrin is usedas the ligand as glioblastoma cells express high levels of thetransferrin receptor on their surface. When melanoma cells are to betargeted, internalizing receptors, monoclonal antibodies directedagainst melanoma-specific surface antigens (e.g., mAb HMSA5) may beemployed as the ligand.

Plasmid DNA encoding polypeptides of interest is formed into a complexwith preformed cationic liposomes using standard methodology oralternatively the DNA is encapsulated into the liposome interior. TheDNA-containing liposomes are then used to transfer the DNA to tumorcells in vivo by direct intra-tumor injection or in vitro (using freshlyexplanted tumor cells) followed by return of the transduced cells to therecipient (e.g., a human patient or non-human animal).

4. Gene Transfer Using Biolistics

Biolistics (microballistics) is a method of delivering DNA into cells byprojection of DNA-coated particles into cells or tissues. DNA is coatedonto the surface of gold or tungsten microparticles (˜1-3 μm diameter)and these particles are accelerated to high velocity and are impactedonto the target cells. The particles burst through the cell membrane andlodge within the target cell. The cell membrane quickly reseals and thepassenger DNA elutes off of the particle and is expressed. The biolisticmethod has been used to transfect mammalian cells [Sanford et al. (1993)Methods Enzymol. 217:483].

A hand-held biolistic apparatus (BioRad) is used to transfer DNA intotumor cells or isolated tumor fragments. This device uses compressedhelium to drive a disc-shaped macroprojectile which carries on itssurface microparticles (1-5 μm) of gold which have been coated withpurified plasmid DNA (coprecipitated with spermine) (Williams et al.,supra). This apparatus has been used to successfully transfect primarytissues.

Plasmid DNA encoding the polypeptides of interest may be coated onto thesurface of gold microparticles according to the manufacturer'sinstructions (BioRad) and the biolistic apparatus is used to transferthe DNA into freshly explanted tumor cells or directly into exposedtumors (e.g., metastatic nodules on the surface of the liver, melanomalesions on the skin).

Regardless of the method of delivery of the expression vector into acell, it is preferred, though not required, that the expression vectorcontain a selection marker (e.g., neo gene) to facilitate selection oftransfected cells. Transfected cells are selected by growth in thepresence of G418 (e.g., 200 μg/ml), followed by culture in growth mediumcontaining reduced concentrations of G418 (e.g., 100 μg/ml) and growthto confluence. Expression of the polypeptides of interest is evaluatedusing, for example, immunoblot analysis or flow cytometry usingmonoclonal antibodies which are specific for the polypeptides ofinterest. It is preferred, though not necessary, that expression of thepolypeptides of interest in the transfected tumor cells is bothconstitutive and stable. Constitutive expression refers to expression inthe absence of a triggering event or condition, and can be achieved bythe selection of a promoter which drives expression of the nucleic acidsequence encoding the polypeptides of interest. Examples of promoterswhich drive constitutive expression of a structural nucleic acidsequence which is operably linked to the promoter include the SRαpromoter, CMV promoter, and HIV promoter.

Regardless of the type of expression vector used for delivery of thenucleic acid sequences of interest into a cell, the expression vectormay be introduced to the cell by direct injection into tumor and/orpreneoplastic tissue, systemic (e.g., intravenous) administration,aerosol administration (e.g., for delivery to the bronchial tree andother lung tissues), injection into breast ducts (e.g., for delivery tobreast tissue), and topical administration (e.g., for delivery tocervical tissue).

F. Reducing Expression of Factor IX in an Animal

The regulatory sequences of the invention may also be used to reduceexpression of a polypeptide sequence of interest which is encoded by anucleic acid sequence whose transcription is under the regulatorycontrol of the regulatory sequences provided herein. For example, theregulatory sequences of the invention may be used to reduce the rate ofage-related increase of FIX activity in an animal as a means of treatingdiseases (e.g., thrombosis, cardiovascular disease, etc.) which areassociated with age-related increases in FIX activity. Since theinventors have discovered that the exemplary nucleic acid sequences AE5′and AE3′ regulate stable and increased expression levels, respectively,of hFIX, the increase in the level of hFIX activity over time may bereduced by inhibiting the function of AE3′ which regulates increasedexpression of hFIX. This approach has the advantage that expression ofhFIX remains under the control of AE5′ thus providing hFIX activitieswhich are stable over time and which continue to play an important rolein normal blood coagulation processes.

The function of AE3′ in age-related expression of FIX may be inhibitedby, for example, inhibiting the activity of the protein whichspecifically binds to AE3′. The protein(s) which bind to AE3′ may beidentified by using the AE3′ (or the minimum portion of AE3′ which hasage-related regulatory activity) to screen protein libraries forspecific binding to AE3′ or its portion. Once the protein which binds toAE3′ is identified, the function of this protein may be inhibited usingantibodies which are specific for this protein. Antibodies which arespecific for the protein which binds to AE3′ are expected to disrupt theinteraction between AE3′ and this protein.

Antibodies (polyclonal and monoclonal) which are specific for theprotein that binds to AE3′ or portions thereof may be generated usingmethods known in the art. The term “antibody” refers to immunoglobulinevoked in animals by an immunogen (antigen). It is desired that theantibody demonstrates specificity to epitopes contained in theimmunogen. The term “polyclonal antibody” refers to immunoglobulinproduced from more than a single clone of plasma cells; in contrast“monoclonal antibody” refers to immunoglobulin produced from a singleclone of plasma cells. The terms “specific binding,” “specificallybinding” and grammatical equivalents thereof when used in reference tothe interaction of an antibody and an immunogen mean that theinteraction is dependent upon the presence of a particular structure(i.e., the antigenic determinant or epitope) on the immunogen; in otherwords the antibody is recognizing and binding to a specific immunogenstructure rather than to immunogens in general. For example, if anantibody is specific for epitope “A”, the presence of an immunogencontaining epitope A (or free, unlabelled A) in a reaction containinglabelled “A” and the antibody will reduce the amount of labelled A boundto the antibody.

Polyclonal and monoclonal antibodies which are specific to a desirablepolypeptide, given the teachings herein, may readily be prepared by oneof skill in the art. For example, monoclonal antibodies may be generatedby immunizing an animal (e.g., mouse, rabbit, etc.) with a desiredantigen and the spleen cells from the immunized animal are immortalized,commonly by fusion with a myeloma cell. Immunization with antigen may beaccomplished in the presence or absence of an adjuvant, e.g., Freund'sadjuvant. Typically, for a mouse, 10 μg antigen in 50-200 μL adjuvant oraqueous solution is administered per mouse by subcutaneous,intraperitoneal or intra-muscular routes. Booster immunization may begiven at intervals, e.g., 2-8 weeks. The final boost is givenapproximately 2-4 days prior to fusion and is generally given in aqueousform rather than in adjuvant.

Spleen cells from the immunized animals may be prepared by teasing thespleen through a sterile sieve into culture medium at room temperature,or by gently releasing the spleen cells into medium by pressure betweenthe frosted ends of two sterile glass microscope slides. The cells areharvested by centrifugation (400 ×g for 5 min.), washed and counted.Spleen cells are fused with myeloma cells to generate hybridoma celllines. Several mouse myeloma cell lines which have been selected forsensitivity to hypoxanthine-aminopterin-thymidine (HAT) are commerciallyavailable and may be grown in, for example, Dulbecco's modified Eagle'smedium (DMEM) (Gibco BRL) containing 10-15% fetal calf serum. Fusion ofmyeloma cells and spleen cells may be accomplished using polyethyleneglycol (PEG) or by electrofusion using protocols which are routine inthe art. Fused cells are distributed into 96-well plates followed byselection of fused cells by culture for 1-2 weeks in 0.1 ml DMEMcontaining 10-15% fetal calf serum and HAT. The supernatants arescreened for antibody production using methods well known in the art.Hybridoma clones from wells containing cells which produce antibody areobtained, e.g., by limiting dilution. Cloned hybridoma cells (4-5×10⁶)are implanted intraperitoneally in recipient mice, preferably of aBALB/c genetic background. Sera and ascites fluids are collected frommice after 10-14 days.

The invention also contemplates humanized antibodies which may begenerated using methods known in the art, such as those described inU.S. Pat. Nos. 5,545,806; 5,569,825 and 5,625,126, the entire contentsos which are incorporated by reference. Such methods include, forexample, generation of transgenic non-human animals which contain humanimmunoglobulin chain genes and which are capable of expressing thesegenes to produce a repertoire of antibodies of various isotypes encodedby the human immunoglobulin genes.

Alternatively, the function of AE3′ in age-related expression of FIX maybe inhibited by, for example, inhibiting the activity of AE3′ usingantisense sequences which are directed to AE3′. The term “antisense” asused herein refers to a deoxyribonucleotide sequence whose sequence ofdeoxyribonucleotide residues is in reverse 5′ to 3′ orientation inrelation to the sequence of deoxyribonucleotide residues in a strand ofa DNA duplex. AE3′ antisense sequences may be used to turn off genesunder the expression regulation of AE3′ by transfecting a cell or tissuewith expression vectors which express high levels of a desired AE3′antisense oligomer (e.g., 15-20 nucleotides) or larger fragment. Suchconstructs can flood cells with antisense sequences which inhibitexpression of FIX. Antisense sequences can be designed from variouslocations along the AE3′ sequence. Animals (e.g., mice) treated withvectors expressing AE3′ antisense sequences are monitored for changes inthe age-related symptoms associated with FIX expression. The alleviationor treatment of one or more of these symptoms in animal by an antisensesequence suggests that the antisense sequence may be useful in thetreatment and/or prevention of age-related FIX expression in humans.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof. Unless otherwise mentioned, all reference tonucleotide numbers with respect to the factor IX nucleotide sequence,refers to the nucleotide numbers of the hFIX gene sequence shown in FIG.8.

EXAMPLE 1 Construction of a Series of Twelve Exemplary Human Factor IX(hFIX) Minigene Expression Vectors

To explore the molecular mechanisms underlying age-related regulation ofFactor IX, a series of twelve hFIX minigene expression vectors wereconstructed. These vectors were first analyzed in vitro in HepG2 cells,a human hepatoma cell line (see Example 2, infra). Transgenic miceharboring the hFIX minigene vectors were generated and longitudinalanalyses of hFIX expression for the entire life spans of founders andsuccessive generations of transgenic mice were carried out (See Example3, infra).

The twelve exemplary minigenes contained sequences derived entirely fromthe hFIX gene sequence, including (a) promoter sequences of variouslengths spanning up to nucleotide (nt)-2231 in the 5′ flanking region,(b) the coding region containing a first intron in which the firstintron's middle portion is truncated. i.e., nt+1098 through nt+5882 ofFIG. 8, and (c) either the complete 3′ UTR sequence or the 3′ UTRsequence in which the middle portion was deleted. FIG. 1 shows thestructure of eleven out of the twelve human FIX minigene expressionconstructs. The name of each construct is shown at left. The structureis depicted with the promoter-containing regions (solid thick line onleft) with the 5′ terminal nucleotide number. Transcribed hFIX regions(open rectangles connected with thin lines representing the shortenedfirst intron) are followed by 3′ flanking sequence regions (solid thickline at right). Arrow: transcription start site; asterisk: translationstop codon; pA: polyadenylation; sl: potential stem-loop formingdinucleotide repeats.

Construction of hFIX minigene expression vectors was carried out using−416FIXm1 as the starting construct (Kurachi et al. (1995) J. Biol.Chem. 270:5276-5281). The nucleotide (nt) numbering system used in thisstudy was based on the complete hFIX gene sequence previously reported(Yoshitake et al. (1985) Biochem. 24:3736-3750). Minigene −416FIXm1/1.4was constructed from −416FIXm1 by inserting the middle portion of the 3′UTR (1.2 kb in size) which was generated by PCR using the followingprimer set with BamH I linkers: 5′ primer,TAACAGGATCCGGCCTCTCACTAACTAATCAC (nt+31418 through +31438) (SEQ IDNO:14) and 3′ primer, CAACTGGATCCAAGATTCAAGATAGAAGGAT (nt+32690 through+32671) (SEQ ID NO:15), and human genomic DNA as an amplificationtemplate. The PCR product was digested with BamH I, and the generatedfragment was inserted into the 3′ UTR BamH I site of −416FIXm1, thusproducing −416FIXm1/1.4 which contained the entire 3′ UTR. −416FIXml/0.7 was constructed by inserting the PCR-amplified 700 bp fragmentwith BamH I linker, containing the 3′ contiguous sequence to nt+32117.The primers used were, 5′ primer: same as that for −416FIXm1/1.4,3′primer: GGACAGGATCCCC CAAACTTTTCAGGCAC (nt+32117 through +32097) (SEQID NO:16). Minigenes −590FIXm1, −679FIXm1, −770FIXm1, −802FIXm1 and−2231FIXm1 were produced by replacing the 5′ end 433 bp sequence of−416FIXm1 released by Sph I/Nhe I digestion with 607, 696, 787, 819 and2248 bp fragments containing the 5′ end hFIX region extended up to nt−590, −679, −770, −802 and −2231, respectively. These latter fragmentswere generated by Sph I/Nhe I digestion of the PCR product obtained with5′ primers: CAAGCATGCATCTAGTGTTAGTGGAAGAG (nt −590 through −571) (SEQ IDNO:17), CAAGCATGCAAATATTAACTCAAAATGGA (nt −679 through −660) (SEQ IDNO:18), CAAGCATGCTGTTGTTTTTTGTTTTAAC (nt −770 through −752) (SEQ IDNO:19), CAAGCATGCAGCCATTCAGTCGAGGAAGG (nt −802 through −783) (SEQ IDNO:20), CAAGCATGCGATCCCTTCCTTATACCT (nt −2231 through −2214) (SEQ IDNO:21) with Sph I linker and the common 3′ primerTAAGCTTAACCTTTGCTAGCAGATTGT (nt+30 through +10) (SEQ ID NO:22) and humangenomic DNA as the amplification template. Minigene −802FIXm1/0.7 (whosestructure is not shown in FIG. 1) contains the 3′ UTR region through nt32,140, which is then connected to nt 32,690 through its downstream poly(A) signal sequence that is common to each of the other elevenconstructs.

−416FIXm1/AE5′ depicts a construct with the AE5′ region moved to the3′-end position and shown as an open box at right. −416FIX ml/AE5′ wasconstructed by inserting the Kpn I fragment generated by PCR (nt −802through nt −417) into the −416FIXm1 vector (the Kpn I site is outside ofthe FIX gene, FIG. 1). The 5′ and 3′ primers used for PCR wereCTTGGTACCAGCCATTCAGTCGAGGAAGG (nt −802 through −783) (SEQ ID NO:23) andCTTGGTACCATATGAATCCTTTCATAGAT, (nt −417 through −436) (SEQ ID NO:24)respectively. All constructs were sequenced through PCR amplifiedregions as well as fragment ligation sites to confirm the correctsequences and orientations.

EXAMPLE 2 Transient Expression of Eleven hFIX Minigene ExpressionVectors In Vitro In Human Hepatoma HepG2 Cell Line

Transient in vitro expression activities of hFIX minigene constructswere assayed using HepG2 cells and hFIX specific enzyme linkedimmunosorbent assay (ELISA) as previously described (Kurachi et al.(1995) J. Biol. Chem. 270:5276-5281) with some modifications. Celltransfection was carried out by the calcium phosphate-DNA conjugatemethod or later, using FuGene 6 (Boehringer Manheim). The latter,improved transfection method consistently increased transfectionefficiency to >20% (Kurachi et al. (1998) Biochemica 3:43-44), and allearlier assays were reexamined using FuGene 6. Four to five independentassays of factor IX activity were carried out and the averages wereshown with standard errors. With FuGene 6 transfection, the controlminigene −416FIXm1 typically produced hFIX at a level of 50 ng/10⁶cells/48 hr.

FIG. 1 shows the relative in vitro transient expression activities ofthe human FIX minigene expression constructs (transient expressionactivity of minigene −802FIXm1/0.7 which is not shown in FIG. 1 was81.5% of the acitity of minigene −416FIXm1). Transient expressionactivities relative to the activity of −416FIXm1 (˜50 ng/10⁶ cells/48hour, and defined as 100% activity) are shown on the right side withstandard deviations (from 4-5 independent assays). Activities werenormalized to the size of minigenes used.

The relative transient expression activities shown in FIG. 1 show thatall constructs showed comparable high transient hFIX expression in HepG2cells (˜50 ng/10⁶ cells/48 hours). However, all the constructscontaining the complete 3′ UTR, including a 102 base pair (bp) stretchof inverted AT, GT and GC dinucleotide repeats [Yoshitake et al. (1985)Biochem. 24:3736-3750], reproducibly showed expression activity levelswhich were 25-30% lower than corresponding minigenes without the repeatsequences. Dinucleotide repeats similar to those seen in the hFIX 3′UTR, which can form stable stem-loop (sl) structures in mRNA, have beenimplicated in controlling mRNA stability in mammals as well as yeast andplants, thus providing an important layer of protein biosynthesisregulation [Ross (1995) Microbiol. Rev. 59:423-450]. Together, theseresults suggest a similar negative regulatory activity for thisstructure of the hFIX gene in the HepG2 assay system on expression ofthe hFIX gene. As described below (e.g., Example 3), however, the 3′ UTRstructure of the hFIX gene containing the dinucleotide repeat regionshowed unexpected functions in vivo which are critical for advancingage-related regulation of the hFIX gene.

Another important and surprising finding with the HepG2 cell assaysystem is that expression by these hFIX minigenes (which containedsequences which are positioned upstream and downstream of the hFIX gene,and which are derived from the homologous hFIX gene instead of fromheterologous reporter genes) does not show any down-regulation in thepresence of the 5′ upstream region (nt −802 up through nt −1900) [Salieret al. (1990) J. Biol. Chem. 265:7062-7068] (FIG. 1). In contrast, whena CAT reporter gene was used, negative regulatory elements wereidentified in this region [Salier et al. (1990) J. Biol. Chem.265:7062-7068].

EXAMPLE 3 Generation And Analysis Of Transgenic Mice Harboring hFIXMinigene Expression Vectors

Transgenic animals were constructed using the expression plasmidsdescribed above in Example 2 according to standard methods [Hogan et al.(1994) in “Manipulating the Mouse Embryo, a Laboratory Manual” (ColdSpring Harbor Press, New York, 2nd Edition). All animal experiments werecarried out in accordance with the institutional guidelines of theUniversity of Michigan (OPRR No. A3114-01).

Briefly, Factor IX minigene expression plasmids were double-digestedwith Sph I/Kpn I and the factor IX minigene-containing fragmentsreleased were isolated by 0.8% agarose gel electrophoresis, followed bypurification with SpinBind DNA extraction units (FMC). Fertilized eggsof C57B/6×SJL mice were microinjected with the DNA (1-2 ng/egg), andimplanted into foster mother animals (CD-1).

A. Multiplex PCR Analysis

Offspring produced were screened for founder animals with high transgenecopy numbers (5-10 copies per genome) using quantitative multiplex PCRanalyses of tail tissue DNA samples. Two pairs of primers were used, onespecific to the hFIX transgenes and the other specific to mouse β-globingene (endogenous control); 5′ primer: CTGTGGGAACACACAGATTTTGG (nt+6172through +6195) (SEQ ID NO:25) and 3′ primer: GGAATAATTCGAATCACAT(nt+30885 through +30867) (SEQ ID NO:26), and 5′ primer:CCAATCTGCTCACACAGGAT (nt+2590 through +2609) (SEQ ID NO:27) and 3′primer: CCTTGAGGCTGTCCAAGTGA (nt+3083 through +3064) (SEQ ID NO:28),respectively. These primers were designed to amplify a unique 966 bpfragment from the hFIX transgenes and a 494 bp fragment from the mouseβ-globin gene, respectively. PCR was initiated with 3 min incubation at94° C., followed by 25 cycles of 94° C. for 30 sec, 65° C. annealing for1 min and 72° C. extension for 2 min.

Founders were back-crossed with non-transgenic mice (C57B/6×SJL) togenerate F1 progeny animals. Homozygous F2 animals were generated bycrossing among heterozygous F1 littermates and the following generationswere similarly generated. Zygosity status of animals was determined byquantitative multiplex PCR analysis as described above. Minimally, threefounder lines for each minigene construct were subjected to longitudinalanalysis for their entire life spans up to two years.

FIG. 3B shows the results of quantitative multiplex PCR analysis todetermine the relative transgene levels in tail and liver tissues.Genomic DNA was extracted from snipped tail tissue of a transgenic−416FIXm1 animal (PA112) at 3 weeks and at 19 months of age. Liver DNAwas extracted from the same animal (PA112) sacrificed at 19 months ofage and a −416FIXm1 animal (PA412) sacrificed at 1 month of age.Positions of hFIX specific fragment (966 bp) and mouse β-globin specificfragment (494 bp, internal copy number control) are shown on the right.Lane 1: kb size ladder; lane 2: fragment size control amplified from−416FIXm1 plasmid; lane 3: non-transgenic mouse tail DNA as template;lane 4: tail DNA of PA112 at 3 weeks of age; lane 5: tail DNA of PA112at 19 months of age; lane 6: liver DNA of PA412 at 1 month of age; lane7: liver DNA of PA112 at 19 month of age. The relative transgene copynumbers for the 1 month-old versus the 19 month-old animals, normalizedto the endogenous mouse β-globin gene, were 1.0-1.1 for both tail aswell as liver genomic DNA preparations, showing no sign of loss of thehFIX transgene in the genome with age (Multi Analyst program from BioRadused for quantitation and calculation of ratios).

B. Immunoassay of hFIX Levels in Transgenic Mice

Circulatory hFIX levels were monitored during longitudinal analyses oftransgenic mice from the representative founder lines carrying varioushFIX minigene transgenes. At various ages, starting at one month of age,transgenic mice were individually subjected to blood sample collection(aliquot of ˜100 μl) via tail-tip snipping, and the obtained serum wasroutinely used to quantify hFIX levels in the circulation usingduplicated hFIX-specific ELISA for each age point. Pooled human plasma(George King Bio-Medical) was used to prepare a hFIX standard curve foreach assay. In order to minimize experimental fluctuations from assay toassay in the longitudinal analysis, overlapped serum samples from theprevious assay group were included in each assay. To ensurereproducibility, three to six independent founder lines were generatedfor each minigene construct, and animals from at least threerepresentative lines were subjected to longitudinal analyses for theirentire life spans. The duplicated ELISA values varied less than 11% fromthe averages. The results are shown in FIGS. 2 and 4.

In all panels in FIGS. 2 and 4, labeling of animals is based on the tagnumbers plus additional information. The first letters of the label F orP represent founder or progeny, respectively. Information on progenygeneration (F1 or F2) and sex are in parenthesis (m: male; f: female),followed by status (+: alive in good health; d: died; s: sacrificed forvarious examinations; mo: age of death or sacrifice). To avoidovercrowding of the panels, the results from representative animals areshown for each minigene construct. Importantly, age-regulation patternswere remarkably similar among all animals for each specific constructand different founder lines. Panels A-E of FIG. 2 show representativefounder line animals with −416FIXm1 (A); −416FIXm1/1.4 (B), −590 FIXm1(C), −679FIXm1 (D) and −770FIXm1 (E). Panels A-D of FIG. 4 showrepresentative founder line animals with −802FIXm1, −802FIXm1/1.4, −2231FIXm1 and −2231FIXm1/1.4, respectively. Panel E shows representativefounder line animals with −416FIXm1/AE5′.

FIG. 2 shows that at one month of age, the mice carrying the −416FIXm1minigene produced hFIX at varying levels, from as high as that ofnatural hFIX gene expression (4 μg/ml) to much lower levels (˜50 ng/ml)(FIG. 2A). Such variations are primarily due to the transgene positionaleffects in the genome. Circulatory hFIX levels of animals from therepresentative founder lines carrying the minigene, however, declineddrastically through puberty and during the subsequent two to three monthperiod to much lower levels, which then remained stable for theremaining life span. This rapid age-dependent characteristic decline inthe circulatory hFIX level was observed in all animals analyzed (n=69),regardless of founder line, differences in initial hFIX level atpre-pubertal age (one month) due to transgene positional effects,generation (founders and F1 or F2 progeny), sex, or zygosity status(homozygous/heterozygous) of the transgenes.

C. Northern Blot Analysis of hFIX mRNA in Transgenic Mice

Northern blot analyses of the liver RNA samples from animals (15 μg perlane) were carried out as previously described [Kurachi et al. (1995)supra] using the ³²P_labeled Ssp I/BamH I fragment (the 3′ half of thehFIX coding region of the cDNA) as a probe, and employing stringentwashing conditions. Under these conditions, the probe preferentiallyhybridized strongly with hFIX minigene mRNA bands (˜1.7 kb) with littlecross-hybridization with the mouse FIX mRNA bands (3.2 kb and 2.2 kb)[Yao et al. (1994) Gene Therapy 1:99-107]. To confirm the presence ofequivalent amounts of RNA in each lane, the filters previouslyhybridized with hFIX probe were stripped of probe and re-probed with theRNR18 cDNA (ribosomal RNA 18S). After completion of longitudinalanalyses of animals from key founder lines for their entire life spans,the representative lines were subjected to embryo-freezing forpreservation.

The results of Northern analysis of human FIX mRNA and transgene DNAlevels in the livers of animals carrying −416FIXm1 are shown in FIG. 3A.hFIX mRNA levels in the liver of young (PA412: F1/f, 1 month of age) andold (PA112; F1/f, 19 months of age) transgenic animals were analyzed byNorthern blot analysis of total liver RNA. PA412 and PA112 animals werefrom the same litter produced by the founder FA661, and expressed 1252and 1675 ng/ml circulatory hFIX at one month of age, respectively. PA112was expressing 63.8 ng/ml serum hFIX at the time of sacrifice. Lane 1:non-transgenic mouse liver RNA; lane 2: transgenic PA412 liver RNA; lane3: transgenic PA112 liver RNA. FIX and 18S on the left or right sidesindicate the band position of hFIXm1 mRNA (˜1.7 kb) and RNR18 (1.9 kb,ribosomal RNA), respectively.

FIG. 3A shows that the decline in blood hFIX level observed in FIG. 2was correlated with a similar decline in the steady-state liver hFIXmRNA, which was not due to a loss of the hFIX transgene with age (FIG.3B). This was further supported by the fact that when 4-5 month old micewith much decreased hFIX levels had progeny, their pups depictedpre-pubertal high hFIX expression levels equivalent to those of theirparents at the same time point (one-month of age).

Minigene vector −416FIXm1/1.4 is identical to −416FIXm1 except that−416FIXm1/1.4 contains the complete 3′UTR, including the dinucleotiderepeat structure (102 bp in length) in its middle region [Yoshitake etal. (1985) Biochem. 24:3736-3750] (FIG. 1). Transgenic mice with−416FIXm1/1.4 (n=48) (FIG. 2B) showed pre-pubertal high and subsequentage-dependent decline in hFIX levels similar to those of −416FIXm1 (FIG.2A), although the decline was less steep and expression levels werestabilized at significantly higher levels than those observed for−416FIXm1 (FIG. 2B).

These results indicate that, while the 102-bp sequence containing thedinucleotide repeat structure of hFIX 3′ UTR reduces the age-relateddecline in expression of hFIX, the presence of the complete 3′ UTRcontaining the extensive dinucleotide repeat structure nonetheless doesnot completely rescue hFIX expression from the age-decline observed inall of these animals, regardless of founder line, initial pre-pubertalhFIX level, generation, sex, or zygosity status of the transgenes.

All animals carrying minigenes −590FIXm1 and −679FIXm1 (a total of 25and 26 animals subjected to longitudinal analysis, respectively) alsoshowed an age-associated rapid decline in hFIX expression similar tothat seen in animals carrying −416FIXm1 (FIG. 2, C and D). Furthermore,hFIX expression levels in three independent founder animals generated todate carrying −770 FIXm1 also rapidly decreased over the puberty periodin a similar pattern as the above minigenes (FIG. 2E). Theseobservations indicated that minigenes with the promoter region up to nt−770 contain the basic structural elements necessary for hFIXexpression, but lack a structural element(s) which functions inage-associated stability of hFIX gene expression.

In contrast, striking and unexpected differences in hFIX expressionpatterns were observed with animals carrying the minigene −802FIXm1(FIG. 4A) as comp[ared to those carrying the minigene −416FIXm1 (FIG.2A). −802FIXm1 is composed of a vector frame identical to −416FIXm1,except that the 5′ end flanking sequence included was extended to nt−802 (FIG. 1).

All animals with −802FIXm1, −2231FIXm1 and −416FIXm1/AE5′ (panels A, C,E) exhibited stable expression throughout their life spans. Animals with−802FIXm1/1.4 and −2231FIXm1/1.4 (FIG. 4 B, D) exhibited age-associatedincreases in hFIX expression levels. All animals maintained or increasedstable circulatory hFIX levels regardless of founder line, initialexpression levels at one month of age, sex, generation or zygositystatus. Mice which died at much younger ages than their normal lifeexpectancies are marked with d. The above results show that all animalsfrom three independent founder lines obtained with −802FIXm1 (FIG. 4A)showed characteristic differences in hFIX expression pattern fromanimals with −416FIXm1 (FIG. 2A) and −416FIXm1/1.4 (FIG. 2B).

The −802FIXm1 transgenic animals (n=62) subjected to longitudinalanalysis invariably showed age-stable plasma hFIX levels for theirentire life spans, mostly up to 20-24 months of age. Age-stablecirculatory hFIX levels were correlated with age-stable mRNA levels(FIG. 5). These observations with −802FIXm1 were further supported byage-stable hFIX expression by mice carrying −2231FIXm1 (FIG. 4C).Together, these results suggest that the structural element which isresponsible for age-stable expression of the hFIX gene resides in thesmall region spanning nt −770 through −802. We designated this smallregion “age-regulatory element in the 5′ end” (AE5′). This regioncontains a transcription factor PEA-3 nucleotide sequence (GAGGAAG: nt−784 through −790), which completely matches the consensus motif(C/G)AGGA(A/T)G [Martin et al. (1988) Proc. Natl. Acad. Sci.85:5839-5843; Xin et al. (1992) Genes & Develop. 6:481-496;Chotteau-Lelievre et al. (1997) Oncogene 15:937-952; Gutman and Wasylyk(1990) EMBO J. 9:2241-2246]. The function of AE5′ nucleotide sequence isposition-independent as shown by age-stable hFIX expression by animalscontaining −416FIXm1/AE5′, in which AE5′ was moved to the 3′ end outsideof the hFIX minigene (FIG. 4E).

Since transgenes of −416FIXm1, −590FIXm1, −679FIXm1 and −770FIXm1 differfrom the minigenes −802FIXm1 and −2231FIXm1 only by their promoters, thehFIX mRNA produced from all of these minigenes (an intron spliced formof FIXm1 RNA) was expected to produce identical hFIX protein. Thus, itwas hypothesized that the age-dependent decline in the circulatory hFIXlevel observed in animals with −416FIXm1, −590FIXm1, −679FIXm1 and−770FIXm1, but not with −802FIXm1 and −2231 FIXm1, must be due to anage-dependent decline in the transcriptional activity of the transgenes.This agrees with the facts that no significant changes with age in hFIXmRNA levels in the liver were observed for animals carrying −802FIXm1(FIG. 5, lanes 2 and 3), while advancing age-dependent declines insteady-state mRNA level were observed for −416FIXm1 (FIG. 3A, lanes 2and 3).

To further determine whether the age-dependent decline in thecirculatory hFIX levels was due to an age-dependent decline intranscriptional activity of the transgenes, the effects of age on hFIXclearance from the circulation were tested as follows. Aliquots ofplasma-derived hFIX preparation (5 μg/0.1 ml of PBS) were injected viatail vein into normal animals at 2, 9-10 and 19-23 months of age (n=3per age group), which have the same genetic background as the transgenicmice (C57B/6×SJL). The hFIX level in circulation was monitored by ELISAof collected blood samples (˜50 μl aliquot) at 10 min, 2, 6, 12, 18, 24,30, 36 and 48 hrs after protein injection. As expected, all animals ofdifferent age groups showed a typical bi-phasic clearance kinetics (twocompartment distribution and clearance) with an initial rapid clearancephase α-phase), followed by a slower clearance phase (β-phase). Theresults are shown in Table 1. TABLE 1 Clearance Time of Human Factor IXin Mice Clearance Time Age (months) (T_(1/2) of Human Factor IX) 2 16.8± 0.21 9-10 17.4 ± 0.55 19-23  16.9 ± 0.35As shown in Table 1, very similar half clearance times were observed forall age groups tested. This agreed with our previous results (17.8hours) for hFIX clearance in a different strain, BALB/c mice (2 monthsof age) (Yao et al. (1994) supra].

Furthermore, the results in Table 1 demonstrate that the hFIX turnovertime from the circulation does not change significantly in vivo withincreasing age, from youth (2 months), to middle age (9-10 months) toold age (19-23 months). These results further confirm that theage-dependent decline in the circulatory hFIX levels was due to anage-dependent decline in transcriptional activity of the transgenes.

It is important to note that in the in vitro HepG2 cell assay system,the presence or absence of AE5′ in the minigenes did not make anysignificant difference in hFIX expression from the hFIX minigenes (FIG.1, and Example 2, supra). In contrast, as mentioned above in thisExample, the presence or absence in vivo of AE5′ makes a dramaticage-dependent difference in hFIX gene expression. This furtherdemonstrates that in vivo longitudinal analysis is important forstudying age-regulation of a gene.

Unlike the animals with −802FIXm1, mice with −802FIXm1/1.4 (whichcontains the complete 3′UTR) showed an advancing age-associated increasein the hFIX level in the circulation (n=48) (FIG. 4 A and B). Thus, todetermine whether this unexpected age-dependent increase in thecirculatory hFIX level was directly correlated with an increased levelof liver hFIX mRNA, Northern blot analyses of transgenic mice carrying−802FIXm1 and −802FIXm1/1.4 were conducted. The hFIX mRNA levels in theliver of 1-month (young) or 15-month (aged) mice carrying −802FIXm1(mouse P327 or P552, respectively) and −802FIXm1/1.4 (mouse P32 andP697, respectively) are shown in FIG. 5. These animals were from thesame litter produced by the founder F17549 for −802FIX ml and F229 for−802FIXm1/1.4 (FIG. 4 A and B). At the time of sacrifice, P552 and P697were expressing 2200 and 1658 ng/ml of hFIX, respectively. The totalliver RNA (15 μg from each animal was used for the Northern blotanalysis performed as described in FIG. 3A. Upper panel: probed with theSsp I/BamH I fragment of hFIX cDNA; lower panel: rehybridized with RNR18(ribosomal RNA) probe. Lane 1: non-transgenic mouse liver; lane 2:transgenic P327 liver RNA; lane 3: transgenic P552 liver RNA; lane 4:transgenic P32 liver RNA; lane 5: transgenic P697 liver RNA.PhosphorImager (Molecular Dynamics) was used for quantitation of mRNAlevels (counts) and ratios of young versus old were calculated. Youngand old animals carrying −802FIXm1 showed no significant differences inthe mRNA level (the ratio of old over young: 0.92). In contrast,−802FIXm1/1.4 animals showed a substantial elevation in the mRNA levelwith older age (the ratio of old over young: 1.54).

These results (FIG. 5, lanes 4 and 5) indicated the presence of anotherimportant age-regulatory nucleotide sequence, designated AE3′, which islocated approximately in the middle of the 3′ UTR where an extensivestretch of dinucleotide repeating structures were contained. In thepresence of AE5′, AE3′ clearly confers a crucial age-associated increasein hFIX expression. This conclusion was further supported by resultsobtained with −2231FIXm1/1.4 (n=42) (FIG. 4D). The unique concertedfunction conferred by the combination of AE5′ and AE3′ was againindependent of founder line, initial expression levels at one month ofage, sex, generation, or zygosity status of animals.

Interestingly, animals with sustained high hFIX levels in thecirculation (approximately 1,500 ng/ml or higher) tended to die at amuch earlier age than the expected life span (˜2 years) (FIG. 4 A, B,D). This happened to both males and females, but appears to be morefrequent in males. Without limiting the invention to any particularmechanism, it is believed that since these transgenic mice have hFIX inaddition to their own mFIX, they may be at an increased risk of lethalthrombosis compared to wild type mice which do not express thetransgenes.

The above-described characterization of transgenic mice harboring hFIXtransgenes demonstrates that (a) while the presence of AE5′ in vitro inHepG2 cells did not affect hFIX gene expression, the presence of AE5′ invivo resulted in a dramatic age-dependent increased stability in hFIXgene expression, (b) the age-dependent decline in the circulatory hFIXlevel observed in animals with −416FIXm1, −590FIXm1, −679FIXm1 and−770FIXm1 is directly correlated with the decrease in the steady-statemRNA level, which the inventors believe to be due to an age-dependentdecline in the transcriptional activity of the transgenes, and (c)animals carrying −802FIXm1/1.4 shwoed a substantial elevation in theliver mRNA levels of hFIX with older age.

EXAMPLE 4 Footprint and Gel Electrophoretic Mobility Shift Analysis ofthe Region from Nucleotides −665 to −805 of Human Factor IX

In order to make a preliminary determination of the region within AE5′which is involved in the function of AE5′, footprint analysis and gelelectrophoretic mobility shift assays were performed as follows.

A. Footprint Analysis

For footprint analysis of the region spanning from nt −665 through nt−805, the fragments used were amplified by PCR with the ³²P-labeled 5′primer ATGGTTAACTGACTTACGAA (nt −833 through −814) (SEQ ID NO:29) and 3′unlabeled primer GCTCCATTTTGAGTTAATATTTGTGT (nt −657 through −682) (SEQID NO:30). The nuclear extracts (NEs) from HepG2 human hepatoma cellsand livers of young (1 month of age) and old (19 months of age) micewere prepared as previously reported [Kurachi et al. (1986) Biochemistry33:1580-1591]. Various amounts of NEs (0, 100 and 150 μg) were incubatedwith the labeled fragments (30,000 CPM) for 1 hour on ice and subjectedto DNase 1 digestion (0.5 unit) for 2 min at room temperature. Thesamples tested included those without NEs, with 100 μg and 150 μg ofHepG2 cell NEs, with 100 μg and 150 μg NEs from old mice, and with 100μg and 150 μg NEs from young mice. Major and minor footprints andapparent DNase hypersensitivity sites were observed.

Footprint analysis of the region nt −665 through −805 with aged mouseliver nuclear extracts showed a major footprint (nt −784 through −802),a minor foot print (nt −721 through −728) and an interesting DNasehypersensitive region (nt −670 through −714). With nuclear extracts fromone month-old animals or HepG2 cells, no such clear footprints wereobserved.

B. Gel Electrophoretic Mobility Shift Assay

Gel electrophoretic mobility shift assay using mouse liver nuclearextracts from three different age groups was used. Nuclear extracts wereprepared from 1, 5 or 19 month-old mouse livers (as described supra).Double stranded oligonucleotides containing a PEA-3 nucleotide sequencespanning from nt −797 to −776 of the hFIX gene (TTCAGTCGAGGAAGGATAGGGT)(SEQ ID NO:31) were ³²P-labeled at the 5′ end to a specific activity of1.9×10⁹ cpm. Aliquots of the radio-labeled oligonucleotide (20,000 cpm)were incubated with 10 μg of NEs in the presence of 1 μg of poly dI-dCin DNA binding buffer for 20 min at room temperature and subjected topolyacrylamide gel electrophoresis (Kurachi et al. (1986) supra). InFIG. 6A, Lane 1: without NEs; lane 2: with NEs of 1 month-old mice; lane3: with NEs of 5 month-old mice; land 4: with NEs of 19 month-old mice;lane 5: with mouse brain NEs (positive control for PEA-3, showing aslightly higher size of shifted band).

FIG. 6B shows the results of the competition assay for ³²P-labeleddouble stranded oligonucleotides containing the PEA-3 nucleotidesequence. A 100-fold excess unlabeled oligonucleotide described in thepreceding paragraph or mutant oligonucleotide [TTCAGTCGGTTGGTGATAGGGT(SEQ ID NO:32) with mutated sequences underlined] was incubated with 10μg of 19 month-old mouse liver NEs for 5 min followed by addition of³²P-labeled oligonucleotides as described supra. Lane 1: without NEs;lane 2: with NEs; lane 3: with NEs and wildtype competitor; lane 4: withNEs and mutant competitor.

In agreement with the above results of footprinting, gel electrophoreticmobility-shift (bandshift) assays showed an increase in protein bindingwith the nuclear extracts of aged mice (19 months of age) (FIG. 6A).Bandshifts were competitively reduced with excess amounts ofoligonucleotides harboring the PEA-3 motif, but not witholigonucleotides harboring a mutant PEA-3 motif sequence (FIG. 6B), thusconfirming the presence of the PEA-3 motif in AE5′. This is the firsttime that the PEA-3 protein, which is a member of the Ets family oftranscription factors and which has been shown to bind to nucleotidesequences [SEQ ID NO:40; SEQ ID NO:48; and SEQ ID NO:84] that arehomologous to the PEA-3 nucleotide sequence within the AE5′ region[Karim et al. (1990) Genes & Develop. 4:1451-1453; Nelsen et al. (1993)Science 261:82-86; Fisher et al. (1991) Oncogene 6:2249-2254], has beenimplicated in such a unique role in age-stable regulation of a gene.

Without limiting the invention to any particular mechanism, the PEA-3nucleotide sequence in the hFIX gene appears to have been generatedthrough evolutionary drift of a L1 sequence originally recruitedpresumably via its retrotransposition into the 5′ specific location.Modern retrotransposable L1 [Kazazian et al. (1988) Nature 332:164-166;Dombroski et al. (1993) Proc. Natl. Acad. Sci. USA 90:6513-6517;Minakami et al. (1992) Nucl. Acids Res. 20:3139-3145; Dombroskiet et al.(1994) Mol. Cell. Biol. 14:4485-4492] does not have the correspondingPEA-3 nucleotide sequence. The PEA-3 nucleotide sequence of AE5′nucleotide sequence resides within the L1-derived sequence retaining a63-70% similarity with the ORF2 corresponding region of the modernretrotransposable L1 in the 5′ to 3′ orientation. Interestingly, themurine FIX gene also has the L1-derived nucleotide sequence in its 5′end region in an almost identical position as in the hFIX gene, and hasmultiple PEA-3 consensus nucleotide elements [Kawarura et al. inOrganization of L1 Sequence in the 5′Flanking Region of Factor IX Gene[in preparation]. Age-regulation of the murine FIX gene is indeed verysimilar to that of the hFIX gene [Sweeney and Hoernig (1993) Am. J.Clin. Pathol. 99:687-688; Kurachi et al. (1996) Thromb. Haemost.76:965-969], thus providing further insights into the evolutionaryorigin of the molecular mechanisms underlying age-associated regulationof the FIX gene.

EXAMPLE 5 Liver-Specific Expression of the Exemplary hFIX Gene underControl of the hFIX Promoter

Expression of the natural FIX gene is virtually restricted to the liver[Salier et al. (1990) J. Biol. Chem. 265:7062-7068]. In order todetermine whether any of the upstream and/or downstream sequences in thehFIX minigenes directed liver-specific expression of the hFIX transgene,Northern blot analysis was carried out as described supra (Example 3) intransgenic mice carrying −416FIXm1 and −802 FIXm1 expression vectors.Animals expressing hFIX at high level (PA412 and P580 carrying −416FIXm1and −802FIXm1, respectively) were sacrificed at one month of age andtotal RNA was extracted from liver, lung, intestine, muscle, kidney,brain and heart and from untransfected HepG2 cells (negative control).The results in transgenic mice carrying −416FIXm1 and −802 FIXm1 areshown in FIGS. 7A and B, respectively.

In FIG. 7, the positions of hFIX mRNA, RNR18 (control for RNA loading inwells), and ribosomal 28S and 18S RNA bands are shown on the left andright sides, respectively. Animals with −416FIXm/1.4 and −679FIXm1showed tissue specific expression patterns similar to that of −416FIXm1(A) (data not shown). Interestingly, liver expression of hFIX observedfor minigenes lacking the region containing AE5′ (except −770FIXm1,which remains to be tested as sufficient progeny animals becomeavailable) was high, but not as robust, as that seen with the naturalgene. In addition, these minigenes expressed not only in the liver, butalso in other tissues, such as kidney, lung and muscle, at variouslevels as high as ˜20% of the liver level (FIG. 7A). In clear contrast,animals with −802FIXm1 showed substantially liver-specific hFIXexpression similar to that for the natural FIX gene (FIG. 7B). Theseresults suggest that the AE5′ region controls liver specific expressionof hFIX.

An apolipoprotein(a) transcription cotnrol region (ACR) whcih containsan ETS family target sequence 5′-CCCGGAAG-3′ (SEQ ID NO:48) has beenshown to exhibit enahncer activity in vitro in liver-derived HepG2cells. However, the ACR does not appear to be liver-specific [Yang etal. (1998), supra].

EXAMPLE 6 Expression of PEA-3 Protein in HepG2 Liver Cells

Expression of the transgene FIX was observed in vivo, but not in vitroin HepG2 cells, when expression vectors containing AE5′ were used (See,Examples 2 and 3, supra). This observation, together with the absence ofa footprint in HepG2 cell NEs (See, Example 4, supra) suggested to theinventors that HepG2 cells' lack of expression of the FIX transgene maybe a result of the cells' expression of low levels of the PEA-3 protein(and/or the PEA-3 related protein) which binds to homologs of theinvention's PEA-3 nucleotide sequence. The complete human PEA-3 cDNA hasnot yet been cloned (the human PEA-3 cDNA sequence of GenBank accessionnumber U18018 lacks 8 amino acids at the N-terminal region when comparedto the mouse PEA-3 cDNA sequence). In order to determine the role of thePEA-3 nucleotide sequence in gene expression in vitro, HepG2 cells whichoverexpress mouse PEA-3 protein were constructed as follows.

Expression constructs containing the mouse PEA-3 cDNA sequence (GenBankAccession Number X63190; FIG. 9) were constructed as follows. Using thereported mouse PEA-3 cDNA sequence three sets of PCR primers weresynthesized such that the entire coding region and parts of the flankingsequences would be amplified. Reverse transcription PCR (RT-PCR) wascarried out, and the amplified mouse PEA-3 cDNA sequence was insertedinto an expression vector under the control of the SV40 promoter, whichdoes not interfere with the factor IX promoter (data not shown).

The PEA-3 expression vector is used to transfect HepG2 cells using theFuGene 6 (Boehringer Manheim) since this method was shown to improvetransfection efficiency (See, Example 2, supra). Transfected HepG2 cellsare screened for expression of PEA-3 by Northern blot analysis and/orWestern blot analysis using commercially available antibody. TransfectedHepG2 cell lines which stably express PEA-3 protein are selected forfurther use, e.g., to analyze the underlying mechanism of PEA-3 action.

EXAMPLE 7 In Vitro and In Vivo Expression of Exemplary Human Protein CMinigene Expression Vectors Containing AE5′ and AE3′

Protein C is a factor which plays a critical role in the anti-bloodcoagulation mechanism. Unlike factor IX, whose level in the circulationsubstantially increases with advancing age, protein C levels in thecirculation do not increase with advancing age, but rather show a slightdecrease over time. This decrease in circulating protein C levels isbelieved by the inventors to be the result of regulation at the genetranscription level. For this reason, the protein C gene provides aninteresting exemplary gene for demonstrating the universality of theAE5′ and AE3′ function in gene expression both in vitro and in vivo asfollows.

A. Construction of Human Protein C Minigene Expression Vectors

The human protein C genomic sequence has been previously reported(GenBank accession number M11228; FIG. 12B]. Using this sequence, threeprotein C minigene expression vectors were prepared. The first humanprotein C minigene vector (−1424PCml) contained the human protein Cpromoter region of the protein C gene (GenBank accession number M11228;FIG. 12 B) ligated to the human protein C cDNA (GenBank accession numberX02750; FIG. 12 A) which contains the first entire intron and poly-Asequence. The second human protein C minigene (AE5′/−1424PCml) was thesame as the first vector vector except that it additionally containedthe nucleotide sequence AE5′ at the 5′ end of the human protein C cDNA.The third human protein C minigene (AE5′/−1424PCml/AE3′) was the same asthe first vector vector except that it additionally contained thenucleotide sequences AE5′ and AE3′ at the 5′ and 3′ ends, respectively,of the human protein C cDNA.

B. Transient Expression of Human Protein C In Vitro in HepG2 Cells

Each of the protein C minigene expression vectors was transfected intoHepG2 cells using the FuGene 6 (Boehringer Mannheim). Four to fiveindependent assays of human protein C activity were carried out aspreviously described [Turkey et al (1999) Throm. Haemost. 81:727-732].HepG2 cells transfected with the −1424PCml vector showed in vitrotransient protein C activities which were comperable to the activitiesshown by HepG2 cells transfected with the AE5′/−1424PCml vector (i.e.,(60-70 ng/10E6 cells/24 hrs).

C. Generation of Transgenic Mice Harboring the Protein C MinigeneExpression Vectors

In order to determine whether AE5′ in combination with AE3′ is capableof increasing human protein C expression with advancing age, as observedfor factor IX expression (Example 3, supra), transgenic mice whichharbor the protein C minigene expression vectors are generated accordingto standard methods [Hogan et al. (1994), supra]. Briefly, protein Cminigene vector plasmids are injected into fertilized eggs of C57B/6×SJLmice and implanted into foster mother animals (CD-1). Offspring producedare screened for founder animals with high transgene copy numbers (5-10copies per genome) using quantitative multiplex PCR analyses of tailtissue DNA samples using two pairs of primers which are designed toamplify a unique fragment from the protein C transgenes and a 494 bpfragment from the mouse β-globin gene, respectively.

Founders are back-crossed with non-transgenic mice (C57B/6×SJL) togenerate F1 progeny animals. Homozygous F2 animals are generated bycrossing among heterozygous F1 littermates and the following generationsare similarly generated. Zygosity status of animals is determined byquantitative multiplex PCR analysis as described above. Founder linesfor each minigene construct are subjected to longitudinal analysis fortheir entire life spans up to two years.

Circulatory human protein C levels are monitored during longitudinalanalyses of transgenic mice from the representative founder linescarrying the protein C minigene transgenes. Age-regulation patterns ofcirculatory human protein C levels are compared among all animals foreach specific construct, different founder lines, different initialhuman protein C level at pre-pubertal age (one month) due to transgenepositional effects, generation (founders and F1 or F2 progeny), sex, andzygosity (homozygous/heterozygous) status of the transgenes.

Northern blot analyses of the liver RNA samples from animals is carriedout using stringent washing conditions to determine whether any changesin circulatory human protein C levels are correlated with similarchanges in the steady-state liver human protein C mRNA, rather than withloss of the human protein C transgene with age or with changes in humanprotein C turnover time. Observation of transgenic animals which containthe human protein C sequence as well as AE5′ and AE3′ and which increasestable circulatory human protein C levels with increasing animal age, ascompared to the levels in transgenic animals which express the humanprotein C sequence in the absence of AE5′ and AE3′, demonstrates thatthe combination of AE5′ and AE3′ functions in age-stable expression ofthe exemplary human protein C gene. This observation will confirm thatthese results may ba achieved for genes other than the exempary hFIX andprotein C genes.

EXAMPLE 8 In Vivo Expression of Exemplary Expression Vectors Containingthe Cytomegalovirus (CMV) Promoter and the AE5′ and AE3′

This Example is carried out to demonstrate the universality of theage-related gene expression regulatory function of AE5′ and AE3′ withviral promoters. The CMV promoter is currently used in several genetherapy constructs but its activity decreases with time in the Liver.Furthermore, the activity of the CMVpromoter in the liver of transgenicmice is known to be lower than the activity in other tissues, such asmuscle. Thus, this Example investigates whether the combination of AE5′and AE3′ halts or reverses the decline in the activity of theCMVpromoter in the liver.

The above-described −416FIXm1 expression vector (Example 1, and FIG. 1)is used to construct a control vector to determine the effect of AE5′and AE3′ on liver and circulatory levels of expression of human factorIX in transgenic animals. The control vector in which expression of thehFIX gene is under the control of the CMV promoter in the absence ofboth AE5′ and AE3′ is constructed by replacing the human factor IXpromoter sequence with the CMV promoter sequence (National Vector Corefor Non-Viral Vectors at the University of Michigan) (the CMV promoteris also located between positions +1 to +596 in vector plasmid pCR3 fromInvitrogen). The resultant expression vector in which the human factorIX gene is under the control of the CMV promoter is transfected intoHepG2 cells. Transfected cells are expected to show human factor IXactivity.

The −802FIXm1/1.4 vector of FIG. 1 is used to construct a test vector inwhich the human factor IX promoter sequence of −802FIXm1/1.4 vector(which contains both AE5′ and AE3′) is replaced with the CMV promotersequence.

In order to determine whether the combination of AE5′ and AE3′ iscapable of increasing human factor IX expression with advancing ageunder the control of the CMV promoter, as observed for factor IXexpression under the control of the factor IX promoter (Example 3,supra), transgenic mice which harbor either the control vector or thetest vector are generated according to standard methods as describedsupra (Examples 3 and 7). The mRNA levels of human factor IX in theblood, liver and other tissues are monitored during longitudinalanalyses of the transgenic mice. Age-regulation patterns of human factorIX mRNA levels in the different tissues are compared among all animalsas described supra for each specific construct, different founder lines,different initial human factor IX levels at pre-pubertal age due totransgene positional effects, generation, sex, and zygosity status ofthe transgenes.

The observation of transgenic animals which contain the test vector andwhich increase stable circulatory human factor IX mRNA levels, ascompared to the circulatory mRNA levels in transgenic animals whichcontain the control vector, demonstrates that the combination of AE5′and AE3′ functions in increasing the activity of the exemplary CMVpromoter.

EXAMPLE 9 Liver-Specific Expression of the Exmplary Human Factor IX Geneunder the Control of the CMV Promoter

This Example investigates whether the presence of AE5′ imparts liverspecific activity to the CMV promoter, which otherwise drives geneexpression in several tissues in addition to the liver.

The −802FIXm1 vector which contains AE5′ and lacks AE3′ (FIG. 1) is usedto construct a test vector in which the human factor IX promotersequence of the −802FIXm1 vector is replaced with the CMV promotersequence. This test vector is used in parallel experiments with thecontrol vector of Example 8 in which expression of the hFIX gene isunder the control of the CMV promoter in the absence of both AE5′ andAE3′. Northern blot analysis is carried out as described supra (Example3) in transgenic mice carrying the control vector or test vector.Animals expressing hFIX at high level are sacrificed at one month of ageand total RNA is extracted from liver, lung, intestine, muscle, kidney,brain and heart and from untransfected HepG2 cells (negative control).The levels of hFIX mRNA in the different tissues are compared. It isexpected that transgenic animals harboring the control vector willexpress hFIX mRNA in liver as well as in at least one other tissue. Incontrast, the observation that transgenic animals which harbor the testvector express hFIX mRNA in the liver and not in other tissues indicatesthat AE5′ confers liver-specific activity to the exemplary CMV promoter.

From the above, it is clear that the invention provides methods forage-related and liver-specific gene expression and models forage-related and liver-specific diseases.

1. A recombinant expression vector comprising in operable combination i)a nucleic acid sequence of interest, ii) a promoter sequence, and iii)one or more age regulatory sequences selected from the group consistingof SEQ ID NO:3 and a functional portion of SEQ ID NO:3.
 2. The vector ofclaim 1, wherein said functional portion of SEQ ID NO:3 is selected fromthe group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ BD NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ IDNO:59, SEQ ID NO:60, and SEQ ID NO:61.
 3. The vector of claim 1, whereinsaid promoter sequence is human factor IX promoter.
 4. The vector ofclaim 1, wherein said nucleic acid sequence of interest encodes factorIX protein.
 5. A recombinant expression vector comprising in operablecombination i) a nucleic acid sequence encoding factor IX protein, ii) ahuman factor IX promoter sequence, and iii) one or more age regulatorysequences selected from the group consisting of SEQ ID NO:3 and afunctional portion of SEQ ID NO:3.
 6. A method of expressing a nucleicacid sequence of interest in a mammalian cell, comprising: a) providing:i) an isolated mammalian cell selected from the group consisting of (a)a liver cell, and (b) a mouse cell selected from the group consisting ofan embryonic stem cell, an eight-cell embryo cell, a blastomere cell, ablastocoele cell, and a fertilized egg cell; ii) a nucleic acid sequenceof interest; iii) a promoter sequence, and iv) one or more ageregulatory sequences selected from SEQ ID NO:3 and a functional portionof SEQ ID NO:3; b) operably linking said nucleic acid sequence ofinterest, said promoter sequence, and said one or more age regulatorysequences to produce a transgene, wherein said age regulatory sequencecomprised in said transgene is located at a position selected from thegroup consisting of the 3′ end of said promoter sequence and the 3′ endof said nucleic acid sequence of interest; and c) introducing saidtransgene into said mammalian cell under conditions such that saidnucleic acid sequence of interest is expressed in said mammalian cell.7. The method of claim 6, wherein said promoter sequence is human factorIX promoter, said nucleic acid sequence of interest encodes factor IXprotein, and said mammalian cell is a liver cell.
 8. The method of claim6, wherein said promoter sequence is human factor IX promoter, saidnucleic acid sequence of interest encodes factor IX protein, and saidmammalian cell is mouse cell selected from the group consisting of anembryonic stem cell, an eight-cell embryo cell, a blastocoele cell, ablastomere cell, a fertilized egg cell, and a liver cell.
 9. The methodof claim 6, wherein said functional portion of SEQ ID NO:3 is selectedfrom the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61.
 10. A method of expressingFactor IX in a mammalian cell, comprising: a) providing: i) an isolatedmammalian cell selected from a liver cell and a mouse cell, said mousecell selected from the group consisting of a fertilized egg cell, anembryonic stem cell, a blastomere cell, a blastocoele cell, and aneight-cell embryo cell; ii) a nucleic acid sequence encoding a Factor IXprotein; iii) a human Factor IX promoter sequence; and iv) one or moreage regulatory sequences selected from the group consisting of SEQ IDNO:3 and a functional portion of SEQ ID NO:3; b) operably linking saidnucleic acid sequence encoding Factor IX protein, said human Factor IXpromoter sequence, and said one or more age regulatory sequences toproduce a transgene, wherein said age regulatory sequence comprised insaid transgene is located at a position selected from the groupconsisting of the 3′ end of said promoter sequence and the 3′ end ofsaid nucleic acid sequence; and c) introducing said transgene into saidmammalian cell under conditions such that said Factor IX protein isexpressed in said mammalian cell.